Human binding molecules capable of neutralizing influenza virus H5N1 and uses thereof

ABSTRACT

Described are binding molecules such as human monoclonal antibodies that bind to influenza virus H5N1 and have neutralizing activity against influenza virus H5N1. Also described are nucleic acid molecules encoding the antibodies, and compositions comprising the antibodies and methods of identifying or producing the antibodies. The antibodies can be used in the diagnosis, prophylaxis, and/or treatment of an influenza virus H5N1 infection. In certain embodiments, the antibodies provide cross-subtype protection in vivo, such that infections with H5, H2, H6, H9, and H1-based influenza subtypes can be prevented and/or treated.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application is a continuation of U.S. patent application Ser. No.13/315,475, filed Dec. 9, 2011, now U.S. Pat. No. 8,691,223 issued Apr.19, 2012, which is a continuation of U.S. patent application Ser. No.12/310,812, filed Mar. 6, 2009, now U.S. Pat. No. 8,192,927, issued Jun.5, 2012, which is a national phase entry under 35 U.S.C. §371 ofInternational Patent Application PCT/EP2007/059356, filed Sep. 6, 2007,published in English as International Patent Publication WO 2008/028946A2 on Mar. 13, 2008, which claims the benefit under Article 8 of thePatent Cooperation Treaty and 35 U.S.C. §119(e) to U.S. ProvisionalPatent Application No. 60/842,930, filed Sep. 7, 2006, and under Article8 of the Patent Cooperation Treaty to European Patent Application Nos.06120316.2 filed Sep. 7, 2006; Ser. No. 06/120,644.7 filed Sep. 14,2006; Ser. No. 06/125,107.0 filed Nov. 30, 2006; and 07111235.3 filedJun. 28, 2007, the disclosure of each of which are hereby incorporatedherein by this reference in their entirety.

STATEMENT ACCORDING TO 37 C.F.R. §1.821(c) or (e) Sequence ListingSubmitted as PDF File with a Request to Transfer CRF from ParentApplication

Pursuant to 37 C.F.R. §1.821(c) or (e), a file containing a PDF versionof the Sequence Listing has been submitted concomitant with thisapplication, the contents of which are hereby incorporated by reference.The transmittal documents of this application include a Request toTransfer CRF from the parent application.

TECHNICAL FIELD

The disclosure relates to biotechnology and medicine. In particular, theinvention relates to the diagnosis, prophylaxis and/or treatment of aninfection by influenza virus H5N1.

BACKGROUND

Influenza viruses consist of three types, A, B, and C. Influenza Aviruses infect a wide variety of birds and mammals, including humans,horses, marine mammals, pigs, ferrets, and chickens. In animals, mostinfluenza A viruses cause mild localized infections of the respiratoryand intestinal tract. However, highly pathogenic influenza A strainssuch as H5N1 exist that cause systemic infections in poultry in whichmortality may reach 100%. Animals infected with influenza A often act asa reservoir for the influenza viruses and certain subtypes have beenshown to cross the species barrier to humans.

Influenza A viruses can be classified into subtypes based on allelicvariations in antigenic regions of two genes that encode surfaceglycoproteins, namely, hemagglutinin (HA) and neuraminidase (NA) whichare required for viral attachment and cellular release. Other majorviral proteins include the nucleoprotein, the nucleocapsid structuralprotein, membrane proteins (M1 and M2), polymerases (PA, PB and PB2) andnon-structural proteins (NS1 and NS2).

Currently, sixteen subtypes of HA (H1-H16) and nine NA (N1-N9) antigenicvariants are known in influenza A virus. Previously, only three subtypeshave been known to circulate in humans (H1N1, H1N2, and H₃N₂). However,in recent years, the pathogenic H5N1 subtype of avian influenza A hasbeen reported to cross the species barrier and infect humans asdocumented in Hong Kong in 1997 and 2003, leading to the death ofseveral patients.

In humans, the avian influenza virus infects cells of the respiratorytract as well as the intestinal tract, liver, spleen, kidneys and otherorgans. Symptoms of avian influenza infection include fever, respiratorydifficulties including shortness of breath and cough, lymphopenia,diarrhea and difficulties regulating blood sugar levels. In contrast toseasonal influenza the group most at risk are healthy adults which makeup the bulk of the population. Due to the high pathogenicity of certainavian influenza A subtypes, particularly H5N1, and their demonstratedability to cross over to infect humans, there is a significant economicand public health risk associated with these viral strains, including areal epidemic and pandemic threat. The scale of the threat isillustrated by the 1918 influenza pandemic which killed over 50 millionpeople.

Currently, no effective vaccines for H5N1 infection are available, sopassive immunotherapy with immunoglobulins may be an alternativestrategy. Use of passive immunization during the 1918 pandemicreportedly halved the death rate.

SUMMARY OF THE INVENTION

Provided are human binding molecules capable of specifically binding toinfluenza virus H5N1 and exhibiting neutralizing activity against H5N1.Also provided are binding molecules that bind to an epitope in thehemagglutinin protein that is shared between influenza subtypes andtherefore relates to binding molecules that cross-react between H5-,H1-, H2-, H6- and H9 influenza based subtypes, such as H5N1, H1N1 andother influenza strains that contain the HA protein with theseparticular epitopes. Further provided are nucleic acid moleculesencoding at least the binding region of the human binding molecules.Still further provided is the use of the human binding molecules in theprophylaxis and/or treatment of a subject having, or at risk ofdeveloping, an H5N1 infection. Besides that, disclosed is the use of thehuman binding molecules in the diagnosis/detection of H5N1. In view oftheir therapeutic benefit in humans, a need exists for bindingmolecules, preferably human binding molecules, able to neutralize H5N1.The disclosure provides these binding molecules and shows that they canbe used in medicine, in particular for diagnosis, prevention and/ortreatment of H5N1 infections.

DESCRIPTION OF THE FIGURES

In FIG. 1 immunoblot analysis of different hemagglutinins (HAS) usingantibodies CR6307 (left part), CR6328 (middle part) and CR511 (rightpart) is presented. Recombinant HAs were subjected to reducing SDS-PAGEanalysis and immunoblot analysis. In lanes 1 sHA of H5N1TV is shown; inlanes 2 recombinant HA, subtype H5 (A/Vietnam/1203/2004 (H5N1)) isshown; in lanes 3 recombinant HA, subtype H3 (A/Wyoming/3/2003(H₃N₂)) isshown; and in lanes 4 recombinant HA, subtype H1 (A/New Calcdonia/20/99(H1N1)) is shown. The position where HA0, HA1 and HA2 can be found isalso indicated.

FIG. 2 shows the average clinical score per group of mice in a study(example 12) wherein, one day before infection with influenza H5N1virus, mice were prophylactically treated with three human H5N1monoclonal antibodies CR6261, CR6323, and CR6325, in different doses.

FIG. 3 shows the change in body weight during the prophylactic treatmentof mice with anti-H5N1 antibodies during 21 days post-infection (example12).

FIG. 4 shows the number of surviving mice in the different groups in thestudy of FIGS. 2 and 3 (Example 12).

FIG. 5 shows the mortality rate in relation to the dose given of theanti-H5N1 antibodies in the study of FIGS. 2-4 (example 12).

FIG. 6 shows the average clinical score per group of mice in a study(example 13) wherein mice were infected with a lethal dose of H5N1influenza virus and treated at different time points after infection (4hr, 1, 2 and 3 days) with CR6261 anti-H5N1 monoclonal antibody, or anon-related antibody CR2006 (administered at day 1 post-infection).

FIG. 7 shows the number of surviving animals in each group of the studydescribed in FIG. 6. All animals of group 1-4, except for one animal ingroup 1 survived the entire study up to day 21 post-infection. Allanimals of group 5 had died at day 9.

FIG. 8 shows the average body weight of the mice in each group duringthe study as described for FIG. 6. Measuring the body weight of the micein group 5 stopped at day 9 as all mice in that group had died by thattime. All remaining mice in groups 1-4 reached normal levels of bodyweight at day 21 post-infection, although it depended on the time oftreatment how fast each group recovered.

FIG. 9 shows the percentage of surviving animals in each group of astudy wherein mice were infected with a lethal dose of H1N1 influenzavirus and treated at different time points (1 day prior-, 1, 2 and 3days post-infection) with CR6261 anti-H5N1 monoclonal antibody, or anon-related antibody CR57 (administered at day 1 post-infection).

FIG. 10 shows the average body weight of the mice in each group duringthe study as described for FIG. 9. Measuring the body weight of the micein group 5 stopped at day 9 as all mice in that group had died by thattime. All remaining mice in groups 1-4 reached normal levels of bodyweight at day 21 post-infection, although it depended on the time oftreatment how fast each group recovered.

DETAILED DESCRIPTION

Here below follow definitions of terms as used herein.

As used herein the term “binding molecule” refers to an intactimmunoglobulin including monoclonal antibodies, such as chimeric,humanized or human monoclonal antibodies, or to an antigen-bindingand/or variable domain comprising fragment of an immunoglobulin thatcompetes with the intact immunoglobulin for specific binding to thebinding partner of the immunoglobulin, e.g., H5N1. Regardless ofstructure, the antigen-binding fragment binds with the same antigen thatis recognized by the intact immunoglobulin. An antigen-binding fragmentcan comprise a peptide or polypeptide comprising an amino acid sequenceof at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100,125, 150, 175, 200, or 250 contiguous amino acid residues of the aminoacid sequence of the binding molecule.

The term “binding molecule,” as used herein includes all immunoglobulinclasses and subclasses known in the art. Depending on the amino acidsequence of the constant domain of their heavy chains, binding moleculescan be divided into the five major classes of intact antibodies: IgA,IgD, IgE, IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4.

Antigen-binding fragments include, inter alia, Fab, F(ab′), F(ab′)2, Fv,dAb, Fd, complementarity determining region (CDR) fragments,single-chain antibodies (scFv), bivalent single-chain antibodies,single-chain phage antibodies, diabodies, triabodies, tetrabodies,(poly)peptides that contain at least a fragment of an immunoglobulinthat is sufficient to confer specific antigen binding to the(poly)peptide, etc. The above fragments may be produced synthetically orby enzymatic or chemical cleavage of intact immunoglobulins or they maybe genetically engineered by recombinant DNA techniques. The methods ofproduction are well known in the art and are described, for example, inAntibodies: A Laboratory Manual, Edited by: E. Harlow and D. Lane(1988), Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., whichis incorporated herein by reference. A binding molecule orantigen-binding fragment thereof may have one or more binding sites. Ifthere is more than one binding site, the binding sites may be identicalto one another or they may be different.

The binding molecule can be a naked or unconjugated binding molecule,but can also be part of an immunoconjugate. A naked or unconjugatedbinding molecule is intended to refer to a binding molecule that is notconjugated, operatively linked or otherwise physically or functionallyassociated with an effector moiety or tag, such as inter alia a toxicsubstance, a radioactive substance, a liposome, an enzyme. It will beunderstood that naked or unconjugated binding molecules do not excludebinding molecules that have been stabilized, multimerized, humanized orin any other way manipulated, other than by the attachment of aneffector moiety or tag. Accordingly, all post-translationally modifiednaked and unconjugated binding molecules are included herewith,including where the modifications are made in the natural bindingmolecule-producing cell environment, by a recombinant bindingmolecule-producing cell, and are introduced by the hand of man afterinitial binding molecule preparation. Of course, the term naked orunconjugated binding molecule does not exclude the ability of thebinding molecule to form functional associations with effector cellsand/or molecules after administration to the body, as some of suchinteractions are necessary in order to exert a biological effect. Thelack of associated effector group or tag is therefore applied indefinition to the naked or unconjugated binding molecule in vitro, notin vivo.

As used herein, the term “biological sample” encompasses a variety ofsample types, including blood and other liquid samples of biologicalorigin, solid tissue samples such as a biopsy specimen or tissuecultures, or cells derived there from and the progeny thereof. The termalso includes samples that have been manipulated in any way after theirprocurement, such as by treatment with reagents, solubilization, orenrichment for certain components, such as proteins or polynucleotides.The term encompasses various kinds of clinical samples obtained from anyspecies, and also includes cells in culture, cell supernatants and celllysates.

The term “complementarity determining regions” (CDR) as used hereinmeans sequences within the variable regions of binding molecules, suchas immunoglobulins, that usually contribute to a large extent to theantigen binding site which is complementary in shape and chargedistribution to the epitope recognized on the antigen. The CDR regionscan be specific for linear epitopes, discontinuous epitopes, orconformational epitopes of proteins or protein fragments, either aspresent on the protein in its native conformation or, in some cases, aspresent on the proteins as denatured, e.g., by solubilization in SDS.Epitopes may also consist of post-translational modifications ofproteins.

The term “deletion,” as used herein, denotes a change in either aminoacid or nucleotide sequence in which one or more amino acid ornucleotide residues, respectively, are absent as compared to the parent,often the naturally occurring, molecule.

The term “expression-regulating nucleic acid sequence” as used hereinrefers to polynucleotide sequences necessary for and/or affecting theexpression of an operably linked coding sequence in a particular hostorganism. The expression-regulating nucleic acid sequences, such asinter alia appropriate transcription initiation, termination, promoter,enhancer sequences; repressor or activator sequences; efficient RNAprocessing signals such as splicing and polyadenylation signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (e.g., ribosome binding sites); sequences thatenhance protein stability; and when desired, sequences that enhanceprotein secretion, can be any nucleic acid sequence showing activity inthe host organism of choice and can be derived from genes encodingproteins, which are either homologous or heterologous to the hostorganism. The identification and employment of expression-regulatingsequences is routine to the person skilled in the art.

The term “functional variant,” as used herein, refers to a bindingmolecule that comprises a nucleotide and/or amino acid sequence that isaltered by one or more nucleotides and/or amino acids compared to thenucleotide and/or amino acid sequences of the parental binding moleculeand that is still capable of competing for binding to the bindingpartner, e.g., H5N1, with the parental binding molecule. In other words,the modifications in the amino acid and/or nucleotide sequence of theparental binding molecule do not significantly affect or alter thebinding characteristics of the binding molecule encoded by thenucleotide sequence or containing the amino acid sequence, i.e. thebinding molecule is still able to recognize and bind its target. Thefunctional variant may have conservative sequence modificationsincluding nucleotide and amino acid substitutions, additions anddeletions. These modifications can be introduced by standard techniquesknown in the art, such as site-directed mutagenesis and randomPCR-mediated mutagenesis, and may comprise natural as well asnon-natural nucleotides and amino acids.

Conservative amino acid substitutions include ones in which the aminoacid residue is replaced with an amino acid residue having similarstructural or chemical properties. Families of amino acid residueshaving similar side chains have been defined in the art. These familiesinclude amino acids with basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., asparagine, glutamine, serine,threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g.,glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine), beta-branched side chains (e.g., threonine, valine,isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine,tryptophan). It will be clear to the skilled artisan that otherclassifications of amino acid residue families than the one used abovecan also be employed. Furthermore, a variant may have non-conservativeamino acid substitutions, e.g., replacement of an amino acid with anamino acid residue having different structural or chemical properties.Similar minor variations may also include amino acid deletions orinsertions, or both. Guidance in determining which amino acid residuesmay be substituted, inserted, or deleted without abolishingimmunological activity may be found using computer programs well knownin the art.

A mutation in a nucleotide sequence can be a single alteration made at alocus (a point mutation), such as transition or transversion mutations,or alternatively, multiple nucleotides may be inserted, deleted orchanged at a single locus. In addition, one or more alterations may bemade at any number of loci within a nucleotide sequence. The mutationsmay be performed by any suitable method known in the art.

The term “host,” as used herein, is intended to refer to an organism ora cell into which a vector such as a cloning vector or an expressionvector has been introduced. The organism or cell can be prokaryotic oreukaryotic. It should be understood that this term is intended to refernot only to the particular subject organism or cell but to the progenyof such an organism or cell as well. Because certain modifications mayoccur in succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentorganism or cell, but are still included within the scope of the term“host” as used herein.

The term “human,” when applied to binding molecules as defined herein,refers to molecules that are either directly derived from a human orbased upon a human sequence. When a binding molecule is derived from orbased on a human sequence and subsequently modified, it is still to beconsidered human as used throughout the specification. In other words,the term human, when applied to binding molecules is intended to includebinding molecules having variable and constant regions derived fromhuman germline immunoglobulin sequences or based on variable or constantregions occurring in a human or human lymphocyte and modified in someform. Thus, the human binding molecules may include amino acid residuesnot encoded by human germline immunoglobulin sequences, comprisesubstitutions and/or deletions (e.g., mutations introduced by forinstance random or site-specific mutagenesis in vitro or by somaticmutation in vivo). “Based on” as used herein refers to the situationthat a nucleic acid sequence may be exactly copied from a template, orwith minor mutations, such as by error-prone PCR methods, orsynthetically made matching the template exactly or with minormodifications. Semi-synthetic molecules based on human sequences arealso considered to be human as used herein.

The term “insertion,” also known as the term “addition,” denotes achange in an amino acid or nucleotide sequence resulting in the additionof one or more amino acid or nucleotide residues, respectively, ascompared to the parent sequence.

The term “isolated,” when applied to binding molecules as definedherein, refers to binding molecules that are substantially free of otherproteins or polypeptides, particularly free of other binding moleculeshaving different antigenic specificities, and are also substantiallyfree of other cellular material and/or chemicals. For example, when thebinding molecules are recombinantly produced, they are preferablysubstantially free of culture medium, and when the binding molecules areproduced by chemical synthesis, they are preferably substantially freeof chemical precursors or other chemicals, i.e., they are separated fromchemical precursors or other chemicals which are involved in thesynthesis of the protein. The term “isolated” when applied to nucleicacid molecules encoding binding molecules as defined herein, is intendedto refer to nucleic acid molecules in which the nucleotide sequencesencoding the binding molecules are free of other nucleotide sequences,particularly nucleotide sequences encoding binding molecules that bindbinding partners other than H5N1. Furthermore, the term “isolated”refers to nucleic acid molecules that are substantially separated fromother cellular components that naturally accompany the native nucleicacid molecule in its natural host, e.g., ribosomes, polymerases, orgenomic sequences with which it is naturally associated. Moreover,“isolated” nucleic acid molecules, such as cDNA molecules, can besubstantially free of other cellular material, or culture medium whenproduced by recombinant techniques, or substantially free of chemicalprecursors or other chemicals when chemically synthesized.

The term “monoclonal antibody” as used herein refers to a preparation ofantibody molecules of single molecular composition. A monoclonalantibody displays a single binding specificity and affinity for aparticular epitope. Accordingly, the term “human monoclonal antibody”refers to an antibody displaying a single binding specificity which hasvariable and constant regions derived from or based on human germlineimmunoglobulin sequences or derived from completely synthetic sequences.The method of preparing the monoclonal antibody is not relevant.

The term “naturally occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organismthat can be isolated from a source in nature and which has not beenintentionally modified by man in the laboratory is naturally occurring.

The term “nucleic acid molecule” as used herein refers to a polymericform of nucleotides and includes both sense and anti-sense strands ofRNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of theabove. A nucleotide refers to a ribonucleotide, deoxynucleotide or amodified form of either type of nucleotide. The term also includessingle- and double-stranded forms of DNA. In addition, a polynucleotidemay include either or both naturally occurring and modified nucleotideslinked together by naturally occurring and/or non-naturally occurringnucleotide linkages. The nucleic acid molecules may be modifiedchemically or biochemically or may contain non-natural or derivatizednucleotide bases, as will be readily appreciated by those of skill inthe art. Such modifications include, for example, labels, methylation,substitution of one or more of the naturally occurring nucleotides withan analog, internucleotide modifications such as uncharged linkages(e.g., methyl phosphonates, phosphotriesters, phosphoramidates,carbamates, etc.), charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), pendent moieties (e.g., polypeptides),intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators,and modified linkages (e.g., alpha anomeric nucleic acids, etc.). Theabove term is also intended to include any topological conformation,including single-stranded, double-stranded, partially duplexed, triplex,hairpinned, circular and padlocked conformations. Also included aresynthetic molecules that mimic polynucleotides in their ability to bindto a designated sequence via hydrogen bonding and other chemicalinteractions. Such molecules are known in the art and include, forexample, those in which peptide linkages substitute for phosphatelinkages in the backbone of the molecule. A reference to a nucleic acidsequence encompasses its complement unless otherwise specified. Thus, areference to a nucleic acid molecule having a particular sequence shouldbe understood to encompass its complementary strand, with itscomplementary sequence. The complementary strand is also useful, e.g.,for anti-sense therapy, hybridization probes and PCR primers.

The term “operably linked” refers to two or more nucleic acid sequenceelements that are usually physically linked and are in a functionalrelationship with each other. For instance, a promoter is operablylinked to a coding sequence, if the promoter is able to initiate orregulate the transcription or expression of a coding sequence, in whichcase the coding sequence should be understood as being “under thecontrol of” the promoter.

By “pharmaceutically acceptable excipient” is meant any inert substancethat is combined with an active molecule such as a drug, agent, orbinding molecule for preparing an agreeable or convenient dosage form.The “pharmaceutically acceptable excipient” is an excipient that isnon-toxic to recipients at the dosages and concentrations employed, andis compatible with other ingredients of the formulation comprising thedrug, agent or binding molecule. Pharmaceutically acceptable excipientsare widely applied in the art.

The term “specifically binding,” as used herein, in reference to theinteraction of a binding molecule, e.g., an antibody, and its bindingpartner, e.g., an antigen, means that the interaction is dependent uponthe presence of a particular structure, e.g., an antigenic determinantor epitope, on the binding partner. In other words, the antibodypreferentially binds or recognizes the binding partner even when thebinding partner is present in a mixture of other molecules or organisms.The binding may be mediated by covalent or non-covalent interactions ora combination of both. In yet other words, the term “specificallybinding” means immunospecifically binding to an antigen or a fragmentthereof and not immunospecifically binding to other antigens. A bindingmolecule that immunospecifically binds to an antigen may bind to otherpeptides or polypeptides with lower affinity as determined by, e.g.,radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA),BIACORE, or other assays known in the art. Binding molecules orfragments thereof that immunospecifically bind to an antigen may becross-reactive with related antigens. Preferably, binding molecules orfragments thereof that immunospecifically bind to an antigen do notcross-react with other antigens.

A “substitution,” as used herein, denotes the replacement of one or moreamino acids or nucleotides by different amino acids or nucleotides,respectively.

The term “therapeutically effective amount” refers to an amount of thebinding molecule as defined herein that is effective for preventing,ameliorating and/or treating a condition resulting from infection withH5N1.

The term “treatment” refers to therapeutic treatment as well asprophylactic or preventative measures to cure or halt or at least retarddisease progress. Those in need of treatment include those alreadyinflicted with a condition resulting from infection with H5N1 as well asthose in which infection with H5N1 is to be prevented. Subjectspartially or totally recovered from infection with H5N1 might also be inneed of treatment. Prevention encompasses inhibiting or reducing thespread of H5N1 or inhibiting or reducing the onset, development orprogression of one or more of the symptoms associated with infectionwith H5N1.

The term “vector” denotes a nucleic acid molecule into which a secondnucleic acid molecule can be inserted for introduction into a host whereit will be replicated, and in some cases expressed. In other words, avector is capable of transporting a nucleic acid molecule to which ithas been linked. Cloning as well as expression vectors are contemplatedby the term “vector,” as used herein. Vectors include, but are notlimited to, plasmids, cosmids, bacterial artificial chromosomes (BAC)and yeast artificial chromosomes (YAC) and vectors derived frombacteriophages or plant or animal (including human) viruses. Vectorscomprise an origin of replication recognized by the proposed host and incase of expression vectors, promoter and other regulatory regionsrecognized by the host. A vector containing a second nucleic acidmolecule is introduced into a cell by transformation, transfection, orby making use of viral entry mechanisms. Certain vectors are capable ofautonomous replication in a host into which they are introduced (e.g.,vectors having a bacterial origin of replication can replicate inbacteria). Other vectors can be integrated into the genome of a hostupon introduction into the host, and thereby are replicated along withthe host genome.

In a first aspect, the disclosure encompasses binding molecules capableof specifically binding to influenza virus A, particularly influenzavirus A subtype H5N1. Preferably, the binding molecules are humanbinding molecules. Preferably, the binding molecules exhibitneutralizing activity against influenza virus A subtype H5N1. In afurther aspect, the binding molecules are able to specifically bind toand/or have neutralizing activity against different influenza virus H5N1strains. These strains may be a member of influenza virus H5N1 strainsof Glade 1, Glade 2 or Glade 3. Phylogenetic analyses of the HA genesfrom the 2004 and 2005 H5N1 outbreak showed two different lineages of HAgenes, termed clades 1 and 2. Viruses in each of these clades aredistributed in non-overlapping geographic regions of Asia. The H5N1viruses from the Indochina peninsula are tightly clustered within Glade1, whereas H5N1 isolates from several surrounding countries are distinctfrom Glade 1 isolates and belong in the more divergent Glade 2. Clade 1H5N1 viruses were isolated from humans and birds in Vietnam, Thailand,and Cambodia, but only from birds in Laos and Malaysia. The Glade 2viruses were found in viruses isolated exclusively from birds in China,Indonesia, Japan, and South Korea. Viruses isolated from birds andhumans in Hong Kong in 2003 and 1997 made up clades 1′ (also categorizedin Glade 1) and 3, respectively (see WHO Global Influenza ProgramSurveillance Network, 2005). The hemagglutinins of the clades differ intheir amino acid sequences. Examples of Glade 1 strains include, but arenot limited to, A/Hong Kong/213/03, ANietnam/1194/04, ANietnam/1203/04and A/Thailand/1(KAN-1)/2004. Examples of Glade 2 strains include, butare not limited to, A/Indonesia/5/05, A/bar headed goose/Qinghai/1A/05,A/turkey/Turkey/1/05 and A/Anhui/1/05. Examples of Glade 3 strainsinclude, but are not limited to, A/Hong Kong/156/97 andA/goose/Guangdong/1/96. Other strains can be found, e.g., in WHO GlobalInfluenza Program Surveillance Network, 2005 and onWorldWideWeb.who.int/csedisease/avian_influenza/guidelines/recommendationvaccine.pdf

The binding molecules may be able to specifically bind to and neutralizeactivity against Glade 1, Glade 2 and Glade 3 strains. The bindingmolecules may be capable of specifically binding to influenza virus H5N1that are viable, living and/or infective or that are ininactivated/attenuated form. Methods for inactivating/attenuatinginfluenza virus H5N1 are well known in the art and include, but are notlimited to, treatment with formalin, (3-propiolactone (BPL),merthiolate, and/or ultraviolet light.

The binding molecules may also be capable of specifically binding to oneor more fragments of influenza virus H5N1 such as inter alia apreparation of one or more proteins and/or (poly)peptides derived fromsubtype H5N1 or one or more recombinantly produced proteins and/orpolypeptides of H5N1. For methods of treatment and/or prevention of H5N1infections, the binding molecules are preferably capable of specificallybinding to surface accessible proteins of H5N1 such as the surfaceglycoproteins, hemagglutinin (HA) and neuraminidase (NA), which arerequired for viral attachment and cellular release, or membrane proteins(M1 and M2). In a specific embodiment, the binding molecules are able tospecifically bind to the HA molecule of H5N1 strains. They may becapable of specifically binding to the HA1 and/or HA2 subunit of the HAmolecule. They may be capable of specifically binding to linear orstructural and/or conformational epitopes on the HA1 and/or HA2 subunitof the HA molecule. The HA molecule may be purified from viruses orrecombinantly produced and optionally isolated before use.Alternatively, HA may be expressed on the surface of cells.

For diagnostic purposes, the binding molecules may also be capable ofspecifically binding to proteins not present on the surface of H5N1including the nucleoprotein, the nucleocapsid structural protein,polymerases (PA, PB and PB2), and non-structural proteins (NS1 and NS2).The nucleotide and/or amino acid molecular sequence of proteins ofvarious H5N1 strains can be found in the GenBank-database, NCBIInfluenza Virus Sequence Database, Influenza Sequence Database (ISD),EMBL-database and/or other databases. It is well within the reach of theskilled person to find such sequences in the respective databases.

In another embodiment, the binding molecules are able to specificallybind to a fragment of the above-mentioned proteins and/or polypeptides,wherein the fragment at least comprises an antigenic determinantrecognized by the binding molecules. An “antigenic determinant” as usedherein is a moiety that is capable of binding to a binding molecule ofthe invention with sufficiently high affinity to form a detectableantigen-binding molecule complex. The binding molecules may or may notbe capable of specifically binding to the extracellular part of HA (alsocalled herein soluble HA (sHA)).

The binding molecules can be intact immunoglobulin molecules such aspolyclonal or monoclonal antibodies or the binding molecules can beantigen-binding fragments including, but not limited to, Fab, F(ab′),F(ab′)₂, Fv, dAb, Fd, complementarity determining region (CDR)fragments, single-chain antibodies (scFv), bivalent single-chainantibodies, single-chain phage antibodies, diabodies, triabodies,tetrabodies, and (poly)peptides that contain at least a fragment of animmunoglobulin that is sufficient to confer specific antigen binding toinfluenza virus H5N1 strains or a fragment thereof. In a preferredembodiment the binding molecules are human monoclonal antibodies.

The binding molecules can be used in non-isolated or isolated form.Furthermore, the binding molecules can be used alone or in a mixturecomprising at least one binding molecule (or variant or fragmentthereof). In other words, the binding molecules can be used incombination, e.g., as a pharmaceutical composition comprising two ormore binding molecules hereof, variants or fragments thereof. Forexample, binding molecules having different, but complementary,activities can be combined in a single therapy to achieve a desiredprophylactic, therapeutic or diagnostic effect, but alternatively,binding molecules having identical activities can also be combined in asingle therapy to achieve a desired prophylactic, therapeutic ordiagnostic effect. Optionally, the mixture further comprises at leastone other therapeutic agent. Preferably, the therapeutic agent such as,e.g., M2 inhibitors (e.g., amantidine, rimantadine) and/or neuraminidaseinhibitors (e.g., zanamivir, oseltamivir) is useful in the prophylaxisand/or treatment of an influenza virus H5N1 infection.

Typically, the binding molecules can bind to their binding partners,i.e. influenza virus H5N1 or fragments thereof, with an affinityconstant (K_(a)-value) that is lower than 0.2×10⁻⁴ M, 1.0×10⁻⁵ M,1.0×10⁻⁶ M, 1.0×10⁻⁷ M, preferably lower than 1.0×10⁻⁸ M, morepreferably lower than 1.0×10⁻⁹ M, more preferably lower than 1.0×10⁻¹°M, even more preferably lower than 1.0×10⁻¹¹ M, and in particular lowerthan 1.0×10⁻¹² M. The affinity constants can vary for antibody isotypes.For example, affinity binding for an IgM isotype refers to a bindingaffinity of at least about 1.0×10⁻⁷ M. Affinity constants can forinstance be measured using surface plasmon resonance, for example usingthe BIACORE system (Pharmacia Biosensor AB, Uppsala, Sweden).

The binding molecules may bind to influenza virus H5N1 or a fragmentthereof in soluble form such as for instance in a sample or insuspension or may bind to influenza virus H5N1 or a fragment thereofbound or attached to a carrier or substrate, e.g., microtiter plates,membranes and beads, etc. Carriers or substrates may be made of glass,plastic (e.g., polystyrene), polysaccharides, nylon, nitrocellulose, orTeflon, etc. The surface of such supports may be solid or porous and ofany convenient shape. Furthermore, the binding molecules may bind toinfluenza virus H5N1 in purified/isolated or non-purified/non-isolatedform.

The binding molecules exhibit neutralizing activity. Neutralizingactivity can for instance be measured as described herein. Alternativeassays measuring neutralizing activity are described in, for instance,WHO Manual on Animal Influenza Diagnosis and Surveillance, Geneva: WorldHealth Organisation, 2005, version 2002.5.

Described is an isolated human binding molecule able to recognize andbind to an epitope in the HA2 subunit of the influenza hemagglutininprotein (HA), characterized in that the binding molecule hasneutralizing activity against an influenza virus comprising HA of the H5subtype. Examples of influenza strains that contain such a HA of the H5subtype and that are important strains in view of pandemic threats areH5N1, H5N2, H5N8, and H5N9. Particularly preferred are binding moleculesthat at least neutralize the H5N1 influenza strain. Preferably, thebinding molecule does not depend on an epitope in the HA1 subunit of theHA protein for binding to the HA protein. The known murine antibody thatwas described in the art (C179) that also binds to the same epitope inthe HA2 domain also depends on binding to an epitope in the HA 1 domainof the HA protein. This is disadvantageous as it adds to the possibilityof escape mutants that are no longer recognized by the antibody.Moreover, a number of the antibodies (such as CR6307 and CR6323) do notdepend on conformational epitopes and recognize the HA2 epitope even ina reduced form (when used in western blotting). This is an advantageover the antibodies from the art because when a conformational change isinduced in the HA protein due to whatever mutation in another part ofthe protein, such conformational change will not most likely hamper thebinding of the antibodies to the HA2 epitope, whereas antibodies that dodepend on conformation might very well be unable to bind when suchmutations occur.

In another preferred embodiment, the binding molecule also hasneutralizing activity against an influenza virus comprising HA of the H1subtype, and preferably wherein the binding molecule also hasneutralizing activity against an influenza virus comprising HA of theH2, H6 and/or H9 subtype. It has been shown herein that the bindingmolecules interact with an epitope present in the HA2 epitopes presentin the H5, H1, H2, H6, and H9 subtypes, and it has been shown that thebinding molecules cross neutralize between influenza subtypes because ofthis epitope-sharing. It is concluded that the binding molecules thatdepend on binding to that particular part in the HA2 domain (and not onanother—mutational prone—epitope in HA1) can cross neutralize betweeninfluenza virus subtypes as they do not appear to depend on binding todomains within HAL which may be altered significantly due to antigenicdrifts. The skilled person, based upon what has been disclosed herein,can now determine whether an antibody indeed cross reacts with HAproteins from different subtypes and also determine whether they areable to neutralize influenza viruses of different subtypes in vivo.

In another aspect, the binding molecule binds to an epitope in the HA2subunit that is selected from the group consisting of amino acidsequence: GVTNKVNSIIDK (SEQ ID NO:368 of the incorporated hereinSequence Listing), GVTNKVNSIIDK (SEQ ID NO:369), GVTNKENSIIDK (SEQ IDNO:370), GVTNKVNRIIDK (SEQ ID NO:371), GITNKVNSVIEK (SEQ ID NO:372),GITNKENSVIEK (SEQ ID NO:373), GITNKVNSIIDK (SEQ ID NO:374), andKITSKVNNIVDK (SEQ ID NO:375). As can be concluded from the data shown inTable 13, certain binding molecules hereof, CR6261, CR6325, and CR6329interact with the GVTNKVNSIIDK (SEQ ID NO:368) epitope present in H5N1,and are not hampered by a mutation in the TGLRN epitope in HA1 that doinfluence the binding of C179. Moreover, some binding molecules, such asCR6307 and CR6323 are not even hampered by a escape mutant, as disclosedin Okuno et al. (1993) with a valine→glutamic acid mutation at position6 (exemplified by GVTNKENSIIDK (SEQ ID NO:370)). This epitope is part ofan extended alpha helix in the HA2 region. The residues in this putativeepitope that are predicted to be most solvent exposed are underlined inbold: GVTNKENSIIDK (SEQ ID NO:370). These amino acids would be mostaccessible to a binding molecule and thus may form the most importantregion of the epitope. Consistent with this notion the highlighted aminoacids are absolutely conserved in identity and position in all thesequences presented. This knowledge could be used to predict bindingepitopes in influenza subtypes that do not carry the same sequence asabove (i.e. H3, H7 and B strains).

Preferred is a binding molecule that is selected from the groupconsisting of:

-   -   a) a binding molecule comprising a heavy chain CDR1 region of        SEQ ID NO:1, a heavy chain CDR2 region of SEQ ID NO:2, and a        heavy chain CDR3 region of SEQ ID NO:3,    -   b) a binding molecule comprising a heavy chain CDR1 region of        SEQ ID NO:16, a heavy chain CDR2 region of SEQ ID NO:17, and a        heavy chain CDR3 region of SEQ ID NO:18,    -   c) a binding molecule comprising a heavy chain CDR1 region of        SEQ ID NO:1, a heavy chain CDR2 region of SEQ ID NO:22, and a        heavy chain CDR3 region of SEQ ID NO:23,    -   d) a binding molecule comprising a heavy chain CDR1 region of        SEQ ID NO:27, a heavy chain CDR2 region of SEQ ID NO:28, and a        heavy chain CDR3 region of SEQ ID NO:29,    -   e) a binding molecule comprising a heavy chain CDR1 region of        SEQ ID NO:39, a heavy chain CDR2 region of SEQ ID NO:40, and a        heavy chain CDR3 region of SEQ ID NO:41,    -   f) a binding molecule comprising a heavy chain CDR1 region of        SEQ ID NO:45, a heavy chain CDR2 region of SEQ ID NO:46, and a        heavy chain CDR3 region of SEQ ID NO:47,    -   g) a binding molecule comprising a heavy chain CDR1 region of        SEQ ID NO:1, a heavy chain CDR2 region of SEQ ID NO:49, and a        heavy chain CDR3 region of SEQ ID NO:50,    -   h) a binding molecule comprising a heavy chain CDR1 region of        SEQ ID NO:52, a heavy chain CDR2 region of SEQ ID NO:53, and a        heavy chain CDR3 region of SEQ ID NO:54,    -   i) a binding molecule comprising a heavy chain CDR1 region of        SEQ ID NO:262, a heavy chain CDR2 region of SEQ ID NO:263, and a        heavy chain CDR3 region of SEQ ID NO:264,    -   j) a binding molecule comprising a heavy chain CDR1 region of        SEQ ID NO:268, a heavy chain CDR2 region of SEQ ID NO:269, and a        heavy chain CDR3 region of SEQ ID NO:270,    -   k) a binding molecule comprising a heavy chain CDR1 region of        SEQ ID NO:274, a heavy chain CDR2 region of SEQ ID NO:275, and a        heavy chain CDR3 region of SEQ ID NO:276,    -   l) a binding molecule comprising a heavy chain CDR1 region of        SEQ ID NO:280, a heavy chain CDR2 region of SEQ ID NO:281, and a        heavy chain CDR3 region of SEQ ID NO:282,    -   m) a binding molecule comprising a heavy chain CDR1 region of        SEQ ID NO:286, a heavy chain CDR2 region of SEQ ID NO:287, and a        heavy chain CDR3 region of SEQ ID NO:288,    -   n) a binding molecule comprising a heavy chain CDR1 region of        SEQ ID NO:292, a heavy chain CDR2 region of SEQ ID NO:293, and a        heavy chain CDR3 region of SEQ ID NO:294,    -   o) a binding molecule comprising a heavy chain CDR1 region of        SEQ ID NO:304, a heavy chain CDR2 region of SEQ ID NO:305, and a        heavy chain CDR3 region of SEQ ID NO:306, and    -   p) a binding molecule comprising a heavy chain CDR1 region of        SEQ ID NO:310, a heavy chain CDR2 region of SEQ ID NO:311, and a        heavy chain CDR3 region of SEQ ID NO:312.

In a preferred embodiment, the binding molecule is for a use as amedicament and preferably for the diagnostic, therapeutic and/orprophylactic treatment of influenza infection. In one aspect of suchuse, the influenza infection is caused by an influenza virus that isassociated with a pandemic outbreak, or has the potential to beassociated with a pandemic outbreak (see, WO 2007/045674 and the tablestherein). Preferably, the influenza virus strain that is associated witha pandemic outbreak and wherein the disease caused by these strains canbe treated by the binding molecules, is selected from the groupconsisting of H1N1, H5N1, H5N2, H5N8, H5N9, H2-based viruses, and H₉N₂.

Also described is a pharmaceutical composition comprising the bindingmolecule, and a pharmaceutically acceptable excipient.

In yet another embodiment, described is the use of a binding molecule inthe preparation of a medicament for the diagnosis, prophylaxis, and/ortreatment of an influenza virus infection. Such infections can occur insmall populations, but can also spread around the world in seasonalepidemics or, worse, in global pandemics where millions of individualsare at risk. Provided are binding molecules that can neutralize theinfection of influenza strains that cause such seasonal epidemics aswell as potential pandemics. Importantly, protection and treatment canbe envisioned now with the binding molecules in relation to multipleinfluenza strains as it has been disclosed that due to the binding of anepitope that is shared between HA proteins of different influenzastrains, cross neutralization between such strains is now possible byusing the binding molecules. This is highly advantageous as the bindingmolecules can protect mammals when they are administered either beforeor after infection (as disclosed in the examples), and therefore alsowhen it is unclear (in early stages of occurrence) whether the infectionis caused by an H1, an H2, an H5, an H6 or an H9-based strain. Healthworkers, military forces as well as the general population may betreated prophylactically, or treated upon infection, with the bindingmolecules. Potentially, the binding molecules can be prepared and storedin huge stocks because it will provide protection against differentpandemic strains, and this will be beneficial in the preparedness ofpossible influenza pandemics in the future.

In a preferred embodiment, the human binding molecules are characterizedin that they are selected from the group consisting of:

-   -   a) a human binding molecule comprising a heavy chain CDR1 region        having the amino acid sequence of SEQ ID NO:1, a heavy chain        CDR2 region having the amino acid sequence of SEQ ID NO:2, a        heavy chain CDR3 region having the amino acid sequence of SEQ ID        NO:3, a light chain CDR1 region having the amino acid sequence        of SEQ ID NO:4, a light chain CDR2 region having the amino acid        sequence of SEQ ID NO:5, and a light chain CDR3 region having        the amino acid sequence of SEQ ID NO:6,    -   b) a human binding molecule comprising a heavy chain CDR1 region        having the amino acid sequence of SEQ ID NO:1, a heavy chain        CDR2 region having the amino acid sequence of SEQ ID NO:2, a        heavy chain CDR3 region having the amino acid sequence of SEQ ID        NO:3, a light chain CDR1 region having the amino acid sequence        of SEQ ID NO:7, a light chain CDR2 region having the amino acid        sequence of SEQ ID NO:8, and a light chain CDR3 region having        the amino acid sequence of SEQ ID NO:9,    -   c) a human binding molecule comprising a heavy chain CDR1 region        having the amino acid sequence of SEQ ID NO:1, a heavy chain        CDR2 region having the amino acid sequence of SEQ ID NO:2, a        heavy chain CDR3 region having the amino acid sequence of SEQ ID        NO:3, a light chain CDR1 region having the amino acid sequence        of SEQ ID NO:10, a light chain CDR2 region having the amino acid        sequence of SEQ ID NO:11, and a light chain CDR3 region having        the amino acid sequence of SEQ ID NO:12,    -   d) a human binding molecule comprising a heavy chain CDR1 region        having the amino acid sequence of SEQ ID NO:1, a heavy chain        CDR2 region having the amino acid sequence of SEQ ID NO:2, a        heavy chain CDR3 region having the amino acid sequence of SEQ ID        NO:3, a light chain CDR1 region having the amino acid sequence        of SEQ ID NO:13, a light chain CDR2 region having the amino acid        sequence of SEQ ID NO:14, and a light chain CDR3 region having        the amino acid sequence of SEQ ID NO:15,    -   e) a human binding molecule comprising a heavy chain CDR1 region        having the amino acid sequence of SEQ ID NO:16, a heavy chain        CDR2 region having the amino acid sequence of SEQ ID NO:17, a        heavy chain CDR3 region having the amino acid sequence of SEQ ID        NO:18, a light chain CDR1 region having the amino acid sequence        of SEQ ID NO:19, a light chain CDR2 region having the amino acid        sequence of SEQ ID NO:20, and a light chain CDR3 region having        the amino acid sequence of SEQ ID NO:21,    -   f) a human binding molecule comprising a heavy chain CDR1 region        having the amino acid sequence of SEQ ID NO:1, a heavy chain        CDR2 region having the amino acid sequence of SEQ ID NO:22, a        heavy chain CDR3 region having the amino acid sequence of SEQ ID        NO:23, a light chain CDR1 region having the amino acid sequence        of SEQ ID NO:24, a light chain CDR2 region having the amino acid        sequence of SEQ ID NO:25, and a light chain CDR3 region having        the amino acid sequence of SEQ ID NO:26,    -   g) a human binding molecule comprising a heavy chain CDR1 region        having the amino acid sequence of SEQ ID NO:27, a heavy chain        CDR2 region having the amino acid sequence of SEQ ID NO:28, a        heavy chain CDR3 region having the amino acid sequence of SEQ ID        NO:29, a light chain CDR1 region having the amino acid sequence        of SEQ ID NO:30, a light chain CDR2 region having the amino acid        sequence of SEQ ID NO:31, and a light chain CDR3 region having        the amino acid sequence of SEQ ID NO:32,    -   h) a human binding molecule comprising a heavy chain CDR1 region        having the amino acid sequence of SEQ ID NO:1, a heavy chain        CDR2 region having the amino acid sequence of SEQ ID NO:2, a        heavy chain CDR3 region having the amino acid sequence of SEQ ID        NO:3, a light chain CDR1 region having the amino acid sequence        of SEQ ID NO:33, a light chain CDR2 region having the amino acid        sequence of SEQ ID NO:34, and a light chain CDR3 region having        the amino acid sequence of SEQ ID NO:35,    -   i) a human binding molecule comprising a heavy chain CDR1 region        having the amino acid sequence of SEQ ID NO:1, a heavy chain        CDR2 region having the amino acid sequence of SEQ ID NO:2, a        heavy chain CDR3 region having the amino acid sequence of SEQ ID        NO:3, a light chain CDR1 region having the amino acid sequence        of SEQ ID NO:36, a light chain CDR2 region having the amino acid        sequence of SEQ ID NO:37, and a light chain CDR3 region having        the amino acid sequence of SEQ ID NO:38,    -   j) a human binding molecule comprising a heavy chain CDR1 region        having the amino acid sequence of SEQ ID NO:39, a heavy chain        CDR2 region having the amino acid sequence of SEQ ID NO:40, a        heavy chain CDR3 region having the amino acid sequence of SEQ ID        NO:41, a light chain CDR1 region having the amino acid sequence        of SEQ ID NO:42, a light chain CDR2 region having the amino acid        sequence of SEQ ID NO:43, and a light chain CDR3 region having        the amino acid sequence of SEQ ID NO:44,    -   k) a human binding molecule comprising a heavy chain CDR1 region        having the amino acid sequence of SEQ ID NO:45, a heavy chain        CDR2 region having the amino acid sequence of SEQ ID NO:46, a        heavy chain CDR3 region having the amino acid sequence of SEQ ID        NO:47, a light chain CDR1 region having the amino acid sequence        of SEQ ID NO:7, a light chain CDR2 region having the amino acid        sequence of SEQ ID NO:8, and a light chain CDR3 region having        the amino acid sequence of SEQ ID NO:48,    -   l) a human binding molecule comprising a heavy chain CDR1 region        having the amino acid sequence of SEQ ID NO:1, a heavy chain        CDR2 region having the amino acid sequence of SEQ ID NO:49, a        heavy chain CDR3 region having the amino acid sequence of SEQ ID        NO:50, a light chain CDR1 region having the amino acid sequence        of SEQ ID NO:33, a light chain CDR2 region having the amino acid        sequence of SEQ ID NO:34, and a light chain CDR3 region having        the amino acid sequence of SEQ ID NO:51,    -   m) a human binding molecule comprising a heavy chain CDR1 region        having the amino acid sequence of SEQ ID NO:52, a heavy chain        CDR2 region having the amino acid sequence of SEQ ID NO:53, a        heavy chain CDR3 region having the amino acid sequence of SEQ ID        NO:54, a light chain CDR1 region having the amino acid sequence        of SEQ ID NO:55, a light chain CDR2 region having the amino acid        sequence of SEQ ID NO:56, and a light chain CDR3 region having        the amino acid sequence of SEQ ID NO:57,    -   n) a human binding molecule comprising a heavy chain CDR1 region        having the amino acid sequence of SEQ ID NO:262, a heavy chain        CDR2 region having the amino acid sequence of SEQ ID NO:263, a        heavy chain CDR3 region having the amino acid sequence of SEQ ID        NO:264, a light chain CDR1 region having the amino acid sequence        of SEQ ID NO:265, a light chain CDR2 region having the amino        acid sequence of SEQ ID NO:266, and a light chain CDR3 region        having the amino acid sequence of SEQ ID NO:267,    -   o) a human binding molecule comprising a heavy chain CDR1 region        having the amino acid sequence of SEQ ID NO:268, a heavy chain        CDR2 region having the amino acid sequence of SEQ ID NO:269, a        heavy chain CDR3 region having the amino acid sequence of SEQ ID        NO:270, a light chain CDR1 region having the amino acid sequence        of SEQ ID NO:271, a light chain CDR2 region having the amino        acid sequence of SEQ ID NO:272, and a light chain CDR3 region        having the amino acid sequence of SEQ ID NO:273,    -   p) a human binding molecule comprising a heavy chain CDR1 region        having the amino acid sequence of SEQ ID NO:274, a heavy chain        CDR2 region having the amino acid sequence of SEQ ID NO:275, a        heavy chain CDR3 region having the amino acid sequence of SEQ ID        NO:276, a light chain CDR1 region having the amino acid sequence        of SEQ ID NO:277, a light chain CDR2 region having the amino        acid sequence of SEQ ID NO:278, and a light chain CDR3 region        having the amino acid sequence of SEQ ID NO:279,    -   q) a human binding molecule comprising a heavy chain CDR1 region        having the amino acid sequence of SEQ ID NO:280, a heavy chain        CDR2 region having the amino acid sequence of SEQ ID NO:281, a        heavy chain CDR3 region having the amino acid sequence of SEQ ID        NO:282, a light chain CDR1 region having the amino acid sequence        of SEQ ID NO:283, a light chain CDR2 region having the amino        acid sequence of SEQ ID NO:284, and a light chain CDR3 region        having the amino acid sequence of SEQ ID NO:285,    -   r) a human binding molecule comprising a heavy chain CDR1 region        having the amino acid sequence of SEQ ID NO:286, a heavy chain        CDR2 region having the amino acid sequence of SEQ ID NO:287, a        heavy chain CDR3 region having the amino acid sequence of SEQ ID        NO:288, a light chain CDR1 region having the amino acid sequence        of SEQ ID NO:289, a light chain CDR2 region having the amino        acid sequence of SEQ ID NO:290, and a light chain CDR3 region        having the amino acid sequence of SEQ ID NO:291,    -   s) a human binding molecule comprising a heavy chain CDR1 region        having the amino acid sequence of SEQ ID NO:292, a heavy chain        CDR2 region having the amino acid sequence of SEQ ID NO:293, a        heavy chain CDR3 region having the amino acid sequence of SEQ ID        NO:294, a light chain CDR1 region having the amino acid sequence        of SEQ ID NO:295, a light chain CDR2 region having the amino        acid sequence of SEQ ID NO:296, and a light chain CDR3 region        having the amino acid sequence of SEQ ID NO:297,    -   t) a human binding molecule comprising a heavy chain CDR1 region        having the amino acid sequence of SEQ ID NO:304, a heavy chain        CDR2 region having the amino acid sequence of SEQ ID NO:305, a        heavy chain CDR3 region having the amino acid sequence of SEQ ID        NO:306, a light chain CDR1 region having the amino acid sequence        of SEQ ID NO:307, a light chain CDR2 region having the amino        acid sequence of SEQ ID NO:308, and a light chain CDR3 region        having the amino acid sequence of SEQ ID NO:309,    -   u) a human binding molecule comprising a heavy chain CDR1 region        having the amino acid sequence of SEQ ID NO:310, a heavy chain        CDR2 region having the amino acid sequence of SEQ ID NO:311, a        heavy chain CDR3 region having the amino acid sequence of SEQ ID        NO:312, a light chain CDR1 region having the amino acid sequence        of SEQ ID NO:313, a light chain CDR2 region having the amino        acid sequence of SEQ ID NO:314, and a light chain CDR3 region        having the amino acid sequence of SEQ ID NO:315,    -   v) a human binding molecule comprising a heavy chain CDR1 region        having the amino acid sequence of SEQ ID NO:238, a heavy chain        CDR2 region having the amino acid sequence of SEQ ID NO:239, a        heavy chain CDR3 region having the amino acid sequence of SEQ ID        NO:240, a light chain CDR1 region having the amino acid sequence        of SEQ ID NO:241, a light chain CDR2 region having the amino        acid sequence of SEQ ID NO:242, and a light chain CDR3 region        having the amino acid sequence of SEQ ID NO:243,    -   w) a human binding molecule comprising a heavy chain CDR1 region        having the amino acid sequence of SEQ ID NO:244, a heavy chain        CDR2 region having the amino acid sequence of SEQ ID NO:245, a        heavy chain CDR3 region having the amino acid sequence of SEQ ID        NO:246, a light chain CDR1 region having the amino acid sequence        of SEQ ID NO:247, a light chain CDR2 region having the amino        acid sequence of SEQ ID NO:248, and a light chain CDR3 region        having the amino acid sequence of SEQ ID NO:249,    -   x) a human binding molecule comprising a heavy chain CDR1 region        having the amino acid sequence of SEQ ID NO:250, a heavy chain        CDR2 region having the amino acid sequence of SEQ ID NO:251, a        heavy chain CDR3 region having the amino acid sequence of SEQ ID        NO:252, a light chain CDR1 region having the amino acid sequence        of SEQ ID NO:253, a light chain CDR2 region having the amino        acid sequence of SEQ ID NO:254, and a light chain CDR3 region        having the amino acid sequence of SEQ ID NO:255,    -   y) a human binding molecule comprising a heavy chain CDR1 region        having the amino acid sequence of SEQ ID NO:256, a heavy chain        CDR2 region having the amino acid sequence of SEQ ID NO:257, a        heavy chain CDR3 region having the amino acid sequence of SEQ ID        NO:258, a light chain CDR1 region having the amino acid sequence        of SEQ ID NO:259, a light chain CDR2 region having the amino        acid sequence of SEQ ID NO:260, and a light chain CDR3 region        having the amino acid sequence of SEQ ID NO:261, and z) a human        binding molecule comprising a heavy chain CDR1 region having the        amino acid sequence of SEQ ID NO:298, a heavy chain CDR2 region        having the amino acid sequence of SEQ ID NO:299, a heavy chain        CDR3 region having the amino acid sequence of SEQ ID NO:300, a        light chain CDR1 region having the amino acid sequence of SEQ ID        NO:301, a light chain CDR2 region having the amino acid sequence        of SEQ ID NO:302, and a light chain CDR3 region having the amino        acid sequence of SEQ ID NO:303.

In a specific embodiment, the binding molecules comprise a heavy chainCDR1 region having the amino acid sequence of SEQ ID NO:1, a heavy chainCDR2 region having the amino acid sequence of SEQ ID NO:2, and a heavychain CDR3 region having the amino acid sequence of SEQ ID NO:3. The CDRregions of the binding molecules are shown in Table 7. CDR regions areaccording to Kabat et al. (1991) as described in Sequences of Proteinsof Immunological Interest. In one embodiment, binding molecules maycomprise two, three, four, five or all six CDR regions as disclosedherein. Preferably, a binding molecule comprises at least two of theCDRs disclosed herein.

In an embodiment, the binding molecules comprise the VH germline VH1-69(see Tomlinson I M, Williams S C, Ignatovitch O, Corbett S J, Winter G.V-BASE Sequence Directory. Cambridge United Kingdom: MRC Centre forProtein Engineering (1997)). In yet another embodiment, the bindingmolecules comprise a heavy chain comprising the variable heavy chain ofthe amino acid sequence selected from the group consisting of SEQ IDNO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ IDNO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ IDNO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:317, SEQ ID NO:321, SEQ IDNO:325, SEQ ID NO:329, SEQ ID NO:333, SEQ ID NO:337, SEQ ID NO:341, SEQID NO:345, SEQ ID NO:349, SEQ ID NO:353, SEQ ID NO:357, SEQ ID NO:361,and SEQ ID NO:365. In a further embodiment, the binding moleculescomprise a light chain comprising the variable light chain of the aminoacid sequence selected from the group consisting of SEQ ID NO:85, SEQ IDNO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ IDNO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ IDNO:107, SEQ ID NO:109, SEQ ID NO:319, SEQ ID NO:323, SEQ ID NO:327, SEQID NO:331, SEQ ID NO:335, SEQ ID NO:339, SEQ ID NO:343, SEQ ID NO:347,SEQ ID NO:351, SEQ ID NO:355, SEQ ID NO:359, SEQ ID NO:363, and SEQ IDNO:367. Table 8 specifies the heavy and light chain variable regions ofthe binding molecule of the invention.

Another aspect includes functional variants of the binding molecules asdefined herein. Molecules are considered to be functional variants of abinding molecule, if the variants are capable of competing forspecifically binding to influenza virus H5N1 or a fragment thereof withthe parental binding molecules. In other words, when the functionalvariants are still capable of binding to influenza virus H5N1 or afragment thereof. Preferably, the functional variants are capable ofcompeting for specifically binding to at least two (or more) differentinfluenza virus H5N1 strains or fragments thereof that are specificallybound by the parental binding molecules. Furthermore, molecules areconsidered to be functional variants of a binding molecule, if they haveneutralizing activity against influenza virus H5N1, preferably againstthe at least two (or more) influenza virus H5N1 strains against whichthe parental binding molecule exhibits neutralizing activity. Functionalvariants include, but are not limited to, derivatives that aresubstantially similar in primary structural sequence, but which contain,e.g., in vitro or in vivo modifications, chemical and/or biochemical,that are not found in the parental binding molecule. Such modificationsinclude inter alia acetylation, acylation, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, cross linking, disulfide bond formation,glycosylation, hydroxylation, methylation, oxidation, pegylation,proteolytic processing, phosphorylation, and the like.

Alternatively, functional variants can be binding molecules as definedherein comprising an amino acid sequence containing substitutions,insertions, deletions or combinations thereof of one or more amino acidscompared to the amino acid sequences of the parental binding molecules.Furthermore, functional variants can comprise truncations of the aminoacid sequence at either or both the amino or carboxyl termini.Functional variants according to the invention may have the same ordifferent, either higher or lower, binding affinities compared to theparental binding molecule but are still capable of binding to influenzavirus H5N1 or a fragment thereof. For instance, functional variantsaccording to the invention may have increased or decreased bindingaffinities for influenza virus H5N1 or a fragment thereof compared tothe parental binding molecules. Preferably, the amino acid sequences ofthe variable regions, including, but not limited to, framework regions,hypervariable regions, in particular the CDR3 regions, are modified.Generally, the light chain and the heavy chain variable regions comprisethree hypervariable regions, comprising three CDRs, and more conservedregions, the so called framework regions (FRs). The hypervariableregions comprise amino acid residues from CDRs and amino acid residuesfrom hypervariable loops. Functional variants intended to fall withinthe scope of the invention have at least about 50% to about 99%,preferably at least about 60% to about 99%, more preferably at leastabout 70% to about 99%, even more preferably at least about 80% to about99%, most preferably at least about 90% to about 99%, in particular atleast about 95% to about 99%, and in particular at least about 97% toabout 99% amino acid sequence homology with the parental bindingmolecules as defined herein. Computer algorithms such as inter alia Gapor Bestfit known to a person skilled in the art can be used to optimallyalign amino acid sequences to be compared and to define similar oridentical amino acid residues. Functional variants can be obtained byaltering the parental binding molecules or parts thereof by generalmolecular biology methods known in the art including, but not limitedto, error prone PCR, oligonucleotide-directed mutagenesis, site directedmutagenesis and heavy and/or light chain shuffling. In an embodiment,the functional variants hereof have neutralizing activity againstinfluenza virus H5N1. The neutralizing activity may either be identical,or be higher or lower compared to the parental binding molecules.Henceforth, when the term (human) binding molecule is used, this alsoencompasses functional variants of the (human) binding molecule.

In yet a further aspect, also described are immunoconjugates, i.e.molecules comprising at least one binding molecule as defined herein andfurther comprising at least one tag, such as inter alia a detectablemoiety/agent. Also contemplated are mixtures of immunoconjugates ormixtures of at least one immunoconjugates according to the invention andanother molecule, such as a therapeutic agent or another bindingmolecule or immunoconjugate. In a further embodiment, theimmunoconjugates may comprise more than one tag. These tags can be thesame or distinct from each other and can be joined/conjugatednon-covalently to the binding molecules. The tag(s) can also bejoined/conjugated directly to the human binding molecules throughcovalent bonding. Alternatively, the tag(s) can be joined/conjugated tothe binding molecules by means of one or more linking compounds.Techniques for conjugating tags to binding molecules are well known tothe skilled artisan.

The tags of the immunoconjugates may be therapeutic agents, but they canalso be detectable moieties/agents. Tags suitable in therapy and/orprevention may be toxins or functional parts thereof, antibiotics,enzymes, other binding molecules that enhance phagocytosis or immunestimulation. Immunoconjugates comprising a detectable agent can be useddiagnostically to, for example, assess if a subject has been infectedwith an influenza virus H5N1 strain or monitor the development orprogression of an influenza virus H5N1 infection as part of a clinicaltesting procedure to, e.g., determine the efficacy of a given treatmentregimen. However, they may also be used for other detection and/oranalytical and/or diagnostic purposes. Detectable moieties/agentsinclude, but are not limited to, enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, bioluminescent materials, radioactivematerials, positron emitting metals, and non-radioactive paramagneticmetal ions. The tags used to label the binding molecules for detectionand/or analytical and/or diagnostic purposes depend on the specificdetection/analysis/diagnosis techniques and/or methods used such asinter alia immunohistochemical staining of (tissue) samples, flowcytometric detection, scanning laser cytometric detection, fluorescentimmunoassays, enzyme linked immunosorbent assays (ELISAs),radioimmunoassays (RIAs), bioassays (e.g., phagocytosis assays), Westernblotting applications, etc. Suitable labels for thedetection/analysis/diagnosis techniques and/or methods known in the artare well within the reach of the skilled artisan.

Furthermore, the human binding molecules or immunoconjugates can also beattached to solid supports, which are particularly useful for in vitroimmunoassays or purification of influenza virus H5N1 or a fragmentthereof. Such solid supports might be porous or nonporous, planar ornon-planar. The binding molecules can be fused to marker sequences, suchas a peptide to facilitate purification. Examples include, but are notlimited to, the hexa-histidine tag, the hemagglutinin (HA) tag, the myctag or the flag tag. Alternatively, an antibody can be conjugated to asecond antibody to form an antibody heteroconjugate. In another aspect,the binding molecules may be conjugated or attached to one or moreantigens. Preferably, these antigens are antigens that are recognized bythe immune system of a subject to which the binding molecule-antigenconjugate is administered. The antigens may be identical, but may alsodiffer from each other. Conjugation methods for attaching the antigensand binding molecules are well known in the art and include, but are notlimited to, the use of cross-linking agents. The binding molecules willbind to influenza virus H5N1 and the antigens attached to the bindingmolecules will initiate a powerful T-cell attack on the conjugate, whichwill eventually lead to the destruction of the influenza virus H5N1.

Next to producing immunoconjugates chemically by conjugating, directlyor indirectly, via for instance a linker, the immunoconjugates can beproduced as fusion proteins comprising the binding molecules and asuitable tag. Fusion proteins can be produced by methods known in theart such as, e.g., recombinantly by constructing nucleic acid moleculescomprising nucleotide sequences encoding the binding molecules in framewith nucleotide sequences encoding the suitable tag(s) and thenexpressing the nucleic acid molecules.

Also provided is a nucleic acid molecule (or “polynucleotide”) encodingat least a binding molecule, functional variant or immunoconjugateaccording to the invention. Such nucleic acid molecules can be used asintermediates for cloning purposes, e.g., in the process of affinitymaturation as described above. In a preferred embodiment, the nucleicacid molecules are isolated or purified.

The skilled person will appreciate that functional variants of thesenucleic acid molecules are also intended to be a part of the invention.Functional variants are nucleic acid sequences that can be directlytranslated, using the standard genetic code, to provide an amino acidsequence identical to that translated from the parental nucleic acidmolecules.

Preferably, the nucleic acid molecules encode binding moleculescomprising the CDR regions as described above. In a further embodimentthe nucleic acid molecules encode binding molecules comprising two,three, four, five or even all six CDR regions of the binding molecules.

In another embodiment, the nucleic acid molecules encode bindingmolecules comprising a heavy chain comprising the variable heavy chainsequences as described above. In another embodiment, the nucleic acidmolecules encode binding molecules comprising a light chain comprisingthe variable light chain sequences as described above. The nucleotidesequences of the binding molecules are given in the Example section(see, e.g., Tables 6 and 8).

Also provided are vectors, e.g., nucleic acid constructs, comprising oneor more nucleic acid molecules according to the invention. Vectors canbe derived from plasmids such as inter alia F, R1, RP1, Col, pBR322,TOL, Ti, etc; cosmids; phages such as lambda, lambdoid, M13, Mu, P1,P22, Qβ, T-even, T-odd, T2, T4, T7, etc; plant viruses. Vectors can beused for cloning and/or for expression of the binding molecules andmight even be used for gene therapy purposes. Vectors comprising one ormore nucleic acid molecules operably linked to one or more expressionregulating nucleic acid molecules are also covered by the disclosure.The choice of the vector is dependent on the recombinant proceduresfollowed and the host used. Introduction of vectors in host cells can beeffected by inter alia calcium phosphate transfection, virus infection,DEAE-dextran mediated transfection, lipofectamine transfection orelectroporation. Vectors may be autonomously replicating or mayreplicate together with the chromosome into which they have beenintegrated. Preferably, the vectors contain one or more selectionmarkers. The choice of the markers may depend on the host cells ofchoice, although this is not critical as is well known to personsskilled in the art. They include, but are not limited to, kanamycin,neomycin, puromycin, hygromycin, ZEOCIN® (phleomycin D1), thymidinekinase gene from Herpes simplex virus (HSV-TK), and dihydrofolatereductase gene from mouse (dhfr) Vectors comprising one or more nucleicacid molecules encoding the human binding molecules as described aboveoperably linked to one or more nucleic acid molecules encoding proteinsor peptides that can be used to isolate the human binding molecules arealso covered by the invention. These proteins or peptides include, butare not limited to, glutathione-S-transferase, maltose binding protein,metal-binding polyhistidine, green fluorescent protein, luciferase andbeta-galactosidase.

Hosts containing one or more copies of the vectors mentioned above arean additional subject of the invention. Preferably, the hosts are hostcells. Host cells include, but are not limited to, cells of mammalian,plant, insect, fungal or bacterial origin. Bacterial cells include, butare not limited to, cells from Gram positive bacteria or Gram negativebacteria such as several species of the genera Escherichia, such as E.coli, and Pseudomonas. In the group of fungal cells preferably yeastcells are used. Expression in yeast can be achieved by using yeaststrains such as inter alia Pichia pastoris, Saccharomyces cerevisiae andHansenula polymorpha. Furthermore, insect cells such as cells fromDrosophila and Sf9 can be used as host cells. Besides that, the hostcells can be plant cells such as inter alia cells from crop plants suchas forestry plants, or cells from plants providing food and rawmaterials such as cereal plants, or medicinal plants, or cells fromornamentals, or cells from flower bulb crops. Transformed (transgenic)plants or plant cells are produced by known methods, for example,Agrobacterium-mediated gene transfer, transformation of leaf discs,protoplast transformation by polyethylene glycol induced DNA transfer,electroporation, sonication, microinjection or bolistic gene transfer.Additionally, a suitable expression system can be a baculovirus system.Expression systems using mammalian cells such as Chinese Hamster Ovary(CHO) cells, COS cells, BHK cells, NSO cells or Bowes melanoma cells arepreferred in the invention. Mammalian cells provide expressed proteinswith post-translational modifications that are most similar to naturalmolecules of mammalian origin. Since the invention deals with moleculesthat may have to be administered to humans, a completely humanexpression system would be particularly preferred. Therefore, even morepreferably, the host cells are human cells. Examples of human cells areinter alia HeLa, 911, AT1080, A549, 293 and HEK293T cells. In certainembodiments, the human producer cells comprise at least a functionalpart of a nucleic acid sequence encoding an adenovirus E1 region inexpressible format. In even more preferred embodiments, the host cellsare derived from a human retina and immortalized with nucleic acidscomprising adenoviral E1 sequences, such as 911 cells or the cell linedeposited at the European Collection of Cell Cultures (ECACC), CAMR,Salisbury, Wiltshire SP4 OJG, Great Britain on 29 Feb. 1996 under number96022940 and marketed under the trademark PER.C6® (PER.C6® is aregistered trademark of Crucell Holland B.V.). For the purposes of thisapplication “PER.C6® cells” refers to cells deposited under number96022940 or ancestors, passages up-stream or downstream as well asdescendants from ancestors of deposited cells, as well as derivatives ofany of the foregoing. Production of recombinant proteins in host cellscan be performed according to methods well known in the art. The use ofthe cells marketed under the trademark PER.C6® as a production platformfor proteins of interest has been described in WO 00/63403, thedisclosure of which is incorporated herein by reference in its entirety.

A method of producing a binding molecule is an additional part hereof.The method comprises the steps of a) culturing a host according to theinvention under conditions conducive to the expression of the bindingmolecule, and b) optionally, recovering the expressed binding molecule.The expressed binding molecules can be recovered from the cell freeextract, but preferably they are recovered from the culture medium. Theabove method of producing can also be used to make functional variantsof the binding molecules and/or immunoconjugates hereof Methods torecover proteins, such as binding molecules, from cell free extracts orculture medium are well known to the man skilled in the art. Bindingmolecules, functional variants and/or immunoconjugates as obtainable bythe above described method are also a part of the invention.

Alternatively, next to the expression in hosts, such as host cells, thebinding molecules and immunoconjugates hereof can be producedsynthetically by conventional peptide synthesizers or in cell freetranslation systems using RNA nucleic acid derived from DNA moleculeshereof Binding molecules and immunoconjugates as obtainable by the abovedescribed synthetic production methods or cell free translation systemsare also a part of the invention.

In yet another embodiment, binding molecules hereof can also be producedin transgenic, non-human, mammals such as inter alia rabbits, goats orcows, and secreted into for instance the milk thereof.

In yet another alternative embodiment, the binding molecules, preferablyhuman binding molecules specifically binding to influenza virus H5N1 ora fragment thereof, may be generated by transgenic non-human mammals,such as for instance transgenic mice or rabbits, which express humanimmunoglobulin genes. Preferably, the transgenic non-human mammals havea genome comprising a human heavy chain transgene and a human lightchain transgene encoding all or a portion of the human binding moleculesas described above. The transgenic non-human mammals can be immunizedwith a purified or enriched preparation of influenza virus H5N1 or afragment thereof. Protocols for immunizing non-human mammals are wellestablished in the art. See Using Antibodies: A Laboratory Manual,Edited by: E. Harlow, D. Lane (1998), Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.; and Current Protocols in Immunology, Editedby: J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W.Strober (2001), John Wiley & Sons Inc., New York, the disclosures ofwhich are incorporated herein by reference Immunization protocols ofteninclude multiple immunizations, either with or without adjuvants such asFreund's complete adjuvant and Freund's incomplete adjuvant, but mayalso include naked DNA immunizations. In another embodiment, the humanbinding molecules are produced by B-cells, plasma and/or memory cellsderived from the transgenic animals. In yet another embodiment, thehuman binding molecules are produced by hybridomas, which are preparedby fusion of B-cells obtained from the above described transgenicnon-human mammals to immortalized cells. B-cells, plasma cells andhybridomas as obtainable from the above described transgenic non-humanmammals and human binding molecules as obtainable from the abovedescribed transgenic non-human mammals, B-cells, plasma and/or memorycells and hybridomas are also a part of the invention.

In a further aspect, provided is a method of identifying a bindingmolecule, such as a human binding molecule, e.g., a human monoclonalantibody or fragment thereof, specifically binding to influenza virusH5N1 or nucleic acid molecules encoding such binding molecules andcomprises the steps of: (a) contacting a collection of binding moleculeson the surface of replicable genetic packages with influenza virus H5N1or a fragment thereof under conditions conducive to binding, (b)selecting at least once for a replicable genetic package binding toinfluenza virus H5N1 or a fragment thereof, (c) separating andrecovering the replicable genetic package binding to influenza virusH5N1 or a fragment thereof from replicable genetic packages that do notbind to influenza virus H5N1 or a fragment thereof. A replicable geneticpackage as used herein can be prokaryotic or eukaryotic and includescells, spores, yeasts, bacteria, viruses, (bacterio)phage, ribosomes andpolysomes. A preferred replicable genetic package is a phage. Thebinding molecules, such as for instance single chain Fvs, are displayedon the replicable genetic package, i.e. they are attached to a group ormolecule located at an exterior surface of the replicable geneticpackage. The replicable genetic package is a screenable unit comprisinga binding molecule to be screened linked to a nucleic acid moleculeencoding the binding molecule. The nucleic acid molecule should bereplicable either in vivo (e.g., as a vector) or in vitro (e.g., by PCR,transcription and translation). In vivo replication can be autonomous(as for a cell), with the assistance of host factors (as for a virus) orwith the assistance of both host and helper virus (as for a phagemid).Replicable genetic packages displaying a collection of binding moleculesis formed by introducing nucleic acid molecules encoding exogenousbinding molecules to be displayed into the genomes of the replicablegenetic packages to form fusion proteins with endogenous proteins thatare normally expressed from the outer surface of the replicable geneticpackages. Expression of the fusion proteins, transport to the outersurface and assembly results in display of exogenous binding moleculesfrom the outer surface of the replicable genetic packages.

The selection step(s) in the method can be performed with influenza H5N1viruses that are live and still infective or inactivated. Inactivationof influenza virus H5N1 may be performed by viral inactivation methodswell known to the skilled artisan such as inter alia treatment withformalin, β-propiolactone (BPL), merthiolate, and/or ultraviolet light.Methods to test, if influenza virus H5N1 is still alive, infectiveand/or viable or partly or completely inactivated are well known to theperson skilled in the art. The influenza virus H5N1 used in the abovemethod may be non-isolated, e.g., present in serum and/or blood of aninfected individual. The influenza virus H5N1 used may also be isolatedfrom cell culture in a suitable medium.

In an embodiment, the influenza virus H5N1 is in suspension whencontacted with the replicable genetic packages. Alternatively, they mayalso be coupled to a carrier when contact takes place. In an embodimenta first and further selection may take place against an influenza virusH5N1 strain of the same Glade. Alternatively, first and furtherselection rounds may be performed against influenza virus H5N1 strainsof different clades (e.g., first selection on Glade 1 strains and secondselection on Glade 2 or 3 strains).

Alternatively, the selection step(s) may be performed in the presence ofa fragment of influenza virus H5N1 such as, e.g., cell membranepreparations, recombinant H5N1 proteins or polypeptides, fusion proteinscomprising H5N1 proteins or polypeptides, cells expressing recombinantH5N1 proteins or polypeptides, and the like. Extracellularly exposedparts of these proteins or polypeptides can also be used as selectionmaterial. The fragments of influenza virus H5N1 may be immobilized to asuitable material before use or may be used in suspension. In anembodiment the selection can be performed on different fragments ofinfluenza virus H5N1 or fragments of different influenza virus H5N1strains. Finding suitable selection combinations are well within thereach of the skilled artisan. Selections may be performed by ELISA orFACS.

In yet a further aspect, provided is a method of obtaining a bindingmolecule specifically binding to an influenza virus H5N1 strain orfragment thereof or a nucleic acid molecule encoding such a bindingmolecule, wherein the method comprises the steps of a) performing theabove described method of identifying binding molecules, and b)isolating from the recovered replicable genetic package the bindingmolecule and/or the nucleic acid molecule encoding the binding molecule.The collection of binding molecules on the surface of replicable geneticpackages can be a collection of scFvs or Fabs. Once a new scFv or Fabhas been established or identified with the above-mentioned method ofidentifying binding molecules or nucleic acid molecules encoding thebinding molecules, the DNA encoding the scFv or Fab can be isolated fromthe bacteria or phages and combined with standard molecular biologicaltechniques to make constructs encoding scFvs, bivalent scFvs, Fabs orcomplete human immunoglobulins of a desired specificity (e.g., IgG, IgAor IgM). These constructs can be transfected into suitable cell linesand complete human monoclonal antibodies can eventually be produced (seeHuls et al., 1999; Boel et al., 2000).

As mentioned before, the preferred replicable genetic package is aphage. Phage display methods for identifying and obtaining (human)binding molecules, e.g. (human) monoclonal antibodies, are by now wellestablished methods known by the person skilled in the art. They are,e.g., described in U.S. Pat. No. 5,696,108; Burton and Barbas, 1994; deKruif et al., 1995b; and Phage Display: A Laboratory Manual. Edited by:C F Barbas, D R Burton, J K Scott and G J Silverman (2001), Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. All these referencesare herewith incorporated herein in their entirety. For the constructionof phage display libraries, collections of human monoclonal antibodyheavy and light chain variable region genes are expressed on the surfaceof bacteriophage, preferably filamentous bacteriophage, particles, in,for example, single chain Fv (scFv) or in Fab format (see de Kruif etal., 1995b). Large libraries of antibody fragment expressing phagestypically contain more than 1.0×10⁹ antibody specificities and may beassembled from the immunoglobulin V-regions expressed in theB-lymphocytes of immunized- or non-immunized individuals. In a specificembodiment, the phage library of binding molecules, preferably scFvphage library, is prepared from RNA isolated from cells obtained from asubject that has been vaccinated against influenza virus (e.g., H5N1),recently vaccinated against an unrelated pathogen, recently sufferedfrom an influenza virus H5N1 infection, or from a healthy individual.RNA can be isolated from inter alia bone marrow or peripheral blood,preferably peripheral blood lymphocytes or isolated B-cells or evensubpopulations of B-cells such as memory B-cells. The subject can be ananimal, preferably a human. In a preferred embodiment, the libraries maybe assembled from the immunoglobulin V-regions expressed by IgM memoryB-cells.

Alternatively, phage display libraries may be constructed fromimmunoglobulin variable regions that have been partially assembled invitro to introduce additional antibody diversity in the library(semi-synthetic libraries). For example, in vitro assembled variableregions contain stretches of synthetically produced, randomized orpartially randomized DNA in those regions of the molecules that areimportant for antibody specificity, e.g., CDR regions. Phage antibodiesspecific for influenza virus H5N1 can be selected from the library byexposing the virus or fragment thereof to a phage library to allowbinding of phages expressing antibody fragments specific for the virusor fragment thereof. Non-bound phages are removed by washing and boundphages eluted for infection of E. Coli bacteria and subsequentpropagation. Multiple rounds of selection and propagation are usuallyrequired to sufficiently enrich for phages binding specifically to thevirus or fragment thereof. If desired, before exposing the phage libraryto the virus or fragment thereof the phage library can first besubtracted by exposing the phage library to non-target material such asviruses or fragments thereof of a different strain, i.e., non-H5N1influenza viruses. These subtractor viruses or fragments thereof can bebound to a solid phase or can be in suspension. Phages may also beselected for binding to complex antigens such as complex mixtures ofH5N1 proteins or (poly)peptides optionally supplemented with othermaterial. Host cells expressing one or more proteins or (poly)peptidesof influenza virus H5N1 may also be used for selection purposes. A phagedisplay method using these host cells can be extended and improved bysubtracting irrelevant binders during screening by addition of an excessof host cells comprising no target molecules or non-target moleculesthat are similar, but not identical, to the target, and thereby stronglyenhance the chance of finding relevant binding molecules. Of course, thesubtraction may be performed before, during or after the screening withvirus or fragments thereof. The process is referred to as the MABSTRACT®process (MABSTRACT® is a registered trademark of Crucell Holland B. V.,see also U.S. Pat. No. 6,265,150, which is incorporated herein byreference).

In yet another aspect, provided is a method of obtaining a bindingmolecule potentially having neutralizing activity against influenzavirus H5N1, preferably at least against influenza virus H5N1 strains ofGlade 1, Glade 2 and Glade 3, wherein the method comprises the steps of(a) performing the method of obtaining a binding molecule specificallybinding to influenza virus H5N1 or a fragment thereof or a nucleic acidmolecule encoding such a binding molecule as described above, and (b)verifying if the binding molecule isolated has neutralizing activityagainst the virus, preferably against at least influenza virus H5N1strains of Glade 1 and Glade 2. Assays for verifying if a bindingmolecule has neutralizing activity are well known in the art (see WHOManual on Animal Influenza Diagnosis and Surveillance, Geneva: WorldHealth Organisation, 2005 version 2002.5).

In a further aspect, provided is a binding molecule having neutralizingactivity being obtainable by the methods as described above.

In yet a further aspect, provided are compositions comprising at least abinding molecule, preferably a human monoclonal antibody, hereof, atleast a functional variant thereof, at least an immunoconjugateaccording to the invention or a combination thereof. In addition tothat, the compositions may comprise inter alia stabilizing molecules,such as albumin or polyethylene glycol, or salts. Preferably, the saltsused are salts that retain the desired biological activity of thebinding molecules and do not impart any undesired toxicological effects.If necessary, the human binding molecules hereof may be coated in or ona material to protect them from the action of acids or other natural ornon-natural conditions that may inactivate the binding molecules.

In yet a further aspect, provided are compositions comprising at least anucleic acid molecule as defined herein. The compositions may compriseaqueous solutions such as aqueous solutions containing salts (e.g., NaClor salts as described above), detergents (e.g., SDS) and/or othersuitable components.

Furthermore, described are pharmaceutical compositions comprising atleast a binding molecule such as a human monoclonal antibody of theinvention (or functional fragment or variant thereof), at least animmunoconjugate according to the invention, at least a compositionaccording to the invention, or combinations thereof. Such apharmaceutical composition further comprises at least onepharmaceutically acceptable excipient. Pharmaceutically acceptableexcipients are well known to the skilled person. The pharmaceuticalcomposition may further comprise at least one other therapeutic agent.Suitable agents are also well known to the skilled artisan.

In an embodiment, the pharmaceutical compositions may comprise two ormore binding molecules that have neutralizing activity against influenzavirus H5N1. In an embodiment, the binding molecules exhibit synergisticneutralizing activity, when used in combination. In other words, thecompositions comprise at least two binding molecules having neutralizingactivity, characterized in that the binding molecules actsynergistically in neutralizing influenza virus H5N1. As used herein,the term “synergistic” means that the combined effect of the bindingmolecules when used in combination is greater than their additiveeffects when used individually. The synergistically acting bindingmolecules may bind to different structures on the same or distinctfragments of influenza virus H5N1. A way of calculating synergy is bymeans of the combination index. The concept of the combination index(CI) has been described by Chou and Talalay (1984). The compositions mayalso comprise one binding molecule having neutralizing activity and onenon-neutralizing H5N1-specific binding molecule. The non-neutralizingand neutralizing H5N1-specific binding molecules may also actsynergistically in neutralizing influenza virus H5N1.

The pharmaceutical composition can further comprise at least one othertherapeutic, prophylactic and/or diagnostic agent. Preferably, thepharmaceutical composition comprises at least one other prophylacticand/or therapeutic agent. Preferably, the further therapeutic and/orprophylactic agents are agents capable of preventing and/or treating aninfluenza virus H5N1 infection and/or a condition resulting from such aninfection. Therapeutic and/or prophylactic agents include, but are notlimited to, anti-viral agents. Such agents can be binding molecules,small molecules, organic or inorganic compounds, enzymes, polynucleotidesequences, anti-viral peptides, etc. Other agents that are currentlyused to treat patients infected with influenza virus H5N1 are M2inhibitors (e.g., amantidine, rimantadine) and/or neuraminidaseinhibitors (e.g., zanamivir, oseltamivir). These can be used incombination with the binding molecules. Agents capable of preventingand/or treating an infection with influenza virus H5N1 and/or acondition resulting from such an infection that are in the experimentalphase might also be used as other therapeutic and/or prophylactic agentsuseful in the invention.

The binding molecules or pharmaceutical compositions hereof can betested in suitable animal model systems prior to use in humans. Suchanimal model systems include, but are not limited to, mouse, ferret andmonkey.

Typically, pharmaceutical compositions must be sterile and stable underthe conditions of manufacture and storage. The binding molecules,immunoconjugates, nucleic acid molecules or compositions hereof can bein powder form for reconstitution in the appropriate pharmaceuticallyacceptable excipient before or at the time of delivery. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-drying(lyophilization) that yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Alternatively, the binding molecules, immunoconjugates, nucleic acidmolecules or compositions hereof can be in solution and the appropriatepharmaceutically acceptable excipient can be added and/or mixed beforeor at the time of delivery to provide a unit dosage injectable form.Preferably, the pharmaceutically acceptable excipient used in theinvention is suitable to high drug concentration, can maintain properfluidity and, if necessary, can delay absorption.

The choice of the optimal route of administration of the pharmaceuticalcompositions will be influenced by several factors including thephysico-chemical properties of the active molecules within thecompositions, the urgency of the clinical situation and the relationshipof the plasma concentrations of the active molecules to the desiredtherapeutic effect. For instance, if necessary, the binding moleculescan be prepared with carriers that will protect them against rapidrelease, such as a controlled release formulation, including implants,transdermal patches, and microencapsulated delivery systems.Biodegradable, biocompatible polymers can inter alia be used, such asethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Furthermore, it may be necessaryto coat the binding molecules with, or co-administer the bindingmolecules with, a material or compound that prevents the inactivation ofthe human binding molecules. For example, the binding molecules may beadministered to a subject in an appropriate carrier, for example,liposomes or a diluent.

The routes of administration can be divided into two main categories,oral and parenteral administration. The preferred administration routeis intravenous or by inhalation.

Oral dosage forms can be formulated inter alia as tablets, troches,lozenges, aqueous or oily suspensions, dispersible powders or granules,emulsions, hard capsules, soft gelatin capsules, syrups or elixirs,pills, dragees, liquids, gels, or slurries. These formulations cancontain pharmaceutically excipients including, but not limited to, inertdiluents, granulating and disintegrating agents, binding agents,lubricating agents, preservatives, coloring, flavoring or sweeteningagents, vegetable or mineral oils, wetting agents, and thickeningagents.

The pharmaceutical compositions hereof can also be formulated forparenteral administration. Formulations for parenteral administrationcan be inter alia in the form of aqueous or non-aqueous isotonic sterilenon-toxic injection or infusion solutions or suspensions. The solutionsor suspensions may comprise agents that are non-toxic to recipients atthe dosages and concentrations employed such as 1,3-butanediol, Ringer'ssolution, Hank's solution, isotonic sodium chloride solution, oils,fatty acids, local anesthetic agents, preservatives, buffers, viscosityor solubility increasing agents, water-soluble antioxidants, oil-solubleantioxidants and metal chelating agents.

In a further aspect, the binding molecules such as human monoclonalantibodies (functional fragments and variants thereof),immunoconjugates, compositions, or pharmaceutical compositions hereofcan be used as a medicament. So, a method of treatment and/or preventionof an influenza virus H5N1 infection using the binding molecules,immunoconjugates, compositions, or pharmaceutical compositions hereof isanother part of the invention. The above-mentioned molecules can interalia be used in the diagnosis, prophylaxis, treatment, or combinationthereof, of an influenza virus H5N1 infection. They are suitable fortreatment of yet untreated patients suffering from an influenza virusH5N1 infection and patients who have been or are treated for aninfluenza virus H5N1 infection. They may be used for patients such ashealthcare workers, relatives of infected subjects, (poultry-)farmers,etc.

These molecules or compositions may be employed in conjunction withother molecules useful in diagnosis, prophylaxis and/or treatment. Theycan be used in vitro, ex vivo or in vivo. For instance, the bindingmolecules such as human monoclonal antibodies (or functional variantsthereof), immunoconjugates, compositions or pharmaceutical compositionshereof can be co-administered with a vaccine against influenza virusH5N1 (if available). Alternatively, the vaccine may also be administeredbefore or after administration of the molecules hereof. Instead of avaccine, anti-viral agents can also be employed in conjunction with thebinding molecules. Suitable anti-viral agents are mentioned above.

The molecules are typically formulated in the compositions andpharmaceutical compositions hereof in a therapeutically ordiagnostically effective amount. Alternatively, they may be formulatedand administered separately. For instance the other molecules such asthe anti-viral agents may be applied systemically, while the bindingmolecules may be applied intravenously.

Dosage regimens can be adjusted to provide the optimum desired response(e.g., a therapeutic response). A suitable dosage range may for instancebe 0.1-100 mg/kg body weight, preferably 0.5-15 mg/kg body weight.Furthermore, for example, a single bolus may be administered, severaldivided doses may be administered over time or the dose may beproportionally reduced or increased as indicated by the exigencies ofthe therapeutic situation. The molecules and compositions according tothe invention are preferably sterile. Methods to render these moleculesand compositions sterile are well known in the art. The other moleculesuseful in diagnosis, prophylaxis and/or treatment can be administered ina similar dosage regimen as proposed for the binding molecules. If theother molecules are administered separately, they may be administered toa patient prior to (e.g., 2 min, 5 min, 10 min, 15 min, 30 min, 45 min,60 min, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 2 days, 3 days,4 days, 5 days, 7 days, 2 weeks, 4 weeks or 6 weeks before),concomitantly with, or subsequent to (e.g., 2 min, 5 min, 10 min, 15min, 30 min, 45 min, 60 min, 2 hours, 4 hours, 6 hours, 8 hours, 10hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24hours, 2 days, 3 days, 4 days, 5 days, 7 days, 2 weeks, 4 weeks or 6weeks after) the administration of one or more of the human bindingmolecules or pharmaceutical compositions hereof. The exact dosingregimen is usually sorted out during clinical trials in human patients.

Human binding molecules and pharmaceutical compositions comprising thehuman binding molecules are particularly useful, and often preferred,when to be administered to human beings as in vivo therapeutic agents,since recipient immune response to the administered antibody will oftenbe substantially less than that occasioned by administration of amonoclonal murine, chimeric or humanized binding molecule.

In another aspect, described is the use of the binding molecules such asneutralizing human monoclonal antibodies (functional fragments andvariants thereof), immunoconjugates, nucleic acid molecules,compositions or pharmaceutical compositions according to the inventionin the preparation of a medicament for the diagnosis, prophylaxis,treatment, or combination thereof, of an influenza virus H5N1 infection.

Kits comprising at least a binding molecule such as a neutralizing humanmonoclonal antibody (functional fragments and variants thereof), atleast an immunoconjugate, at least a nucleic acid molecule, at least acomposition, at least a pharmaceutical composition, at least a vector,at least a host hereof or a combination thereof are also a part hereofOptionally, the above-described components of the kits hereof are packedin suitable containers and labeled for diagnosis, prophylaxis and/ortreatment of the indicated conditions. The above-mentioned componentsmay be stored in unit or multi-dose containers as an aqueous, preferablysterile, solution or as a lyophilized, preferably sterile, formulationfor reconstitution. The containers may be formed from a variety ofmaterials such as glass or plastic and may have a sterile access port(for example, the container may be an intravenous solution bag or a vialhaving a stopper pierceable by a hypodermic injection needle). The kitmay further comprise more containers comprising a pharmaceuticallyacceptable buffer. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, syringes, culture medium for one or more of thesuitable hosts and, possibly, even at least one other therapeutic,prophylactic or diagnostic agent. Associated with the kits can beinstructions customarily included in commercial packages of therapeutic,prophylactic or diagnostic products, that contain information about forexample the indications, usage, dosage, manufacture, administration,contra-indications and/or warnings concerning the use of suchtherapeutic, prophylactic or diagnostic products.

Further described is a method of detecting influenza virus H5N1 in asample, wherein the method comprises the steps of (a) contacting asample with a diagnostically effective amount of a binding molecule(functional fragments and variants thereof) or an immunoconjugateaccording to the invention, and (b) determining whether the bindingmolecule or immunoconjugate specifically binds to a molecule of thesample. The sample may be a biological sample including, but not limitedto blood, serum, stool, sputum, nasopharyngeal aspirates, bronchiallavages, urine, tissue or other biological material from (potentially)infected subjects, or a non-biological sample such as water, drink, etc.The (potentially) infected subjects may be human subjects, but alsoanimals that are suspected as carriers of influenza virus H5N1 might betested for the presence of the virus using the human binding moleculesor immunoconjugates hereof. The sample may first be manipulated to makeit more suitable for the method of detection. Manipulation means interalia treating the sample suspected to contain and/or containing thevirus in such a way that the virus will disintegrate into antigeniccomponents such as proteins, (poly)peptides or other antigenicfragments. Preferably, the human binding molecules or immunoconjugateshereof are contacted with the sample under conditions which allow theformation of an immunological complex between the human bindingmolecules and the virus or antigenic components thereof that may bepresent in the sample. The formation of an immunological complex, ifany, indicating the presence of the virus in the sample, is thendetected and measured by suitable means. Such methods include, interalia, homogeneous and heterogeneous binding immunoassays, such asradio-immunoassays (RIA), ELISA, immunofluorescence,immunohistochemistry, FACS, BIACORE and Western blot analyses.

Preferred assay techniques, especially for large-scale clinicalscreening of patient sera and blood and blood-derived products are ELISAand Western blot techniques. ELISA tests are particularly preferred. Foruse as reagents in these assays, the binding molecules orimmunoconjugates hereof are conveniently bonded to the inside surface ofmicrotiter wells. The binding molecules or immunoconjugates hereof maybe directly bonded to the microtiter well. However, maximum binding ofthe binding molecules or immunoconjugates hereof to the wells might beaccomplished by pre-treating the wells with polylysine prior to theaddition of the binding molecules or immunoconjugates hereof.Furthermore, the binding molecules or immunoconjugates hereof may becovalently attached by known means to the wells. Generally, the bindingmolecules or immunoconjugates are used between 0.01 to 100 μg/ml forcoating, although higher as well as lower amounts may also be used.Samples are then added to the wells coated with the binding molecules orimmunoconjugates hereof

Furthermore, binding molecules hereof can be used to identify specificbinding structures of influenza virus H5N1. The binding structures canbe epitopes on proteins and/or polypeptides. They can be linear, butalso structural and/or conformational. In one embodiment, the bindingstructures can be analyzed by means of PEPSCAN analysis (see, interalia, WO 84/03564, WO 93/09872, Slootstra et al., 1996). Alternatively,a random peptide library comprising peptides from a protein of influenzavirus H5N1 can be screened for peptides capable of binding to thebinding molecules. The binding structures/peptides/epitopes found can beused as vaccines and for the diagnosis of influenza virus H5N1infections. In case fragments other than proteins and/or polypeptidesare bound by the binding molecules, binding structures can be identifiedby mass spectrometry, high performance liquid chromatography and nuclearmagnetic resonance.

Also provided is a method of screening a binding molecule (or afunctional fragment or variant thereof) for specific binding to the sameepitope of influenza virus H5N1, as the epitope bound by a human bindingmolecule hereof, wherein the method comprises (a) contacting a bindingmolecule to be screened, a binding molecule hereof and influenza virusH5N1 or a fragment thereof, (b) measure if the binding molecule to bescreened is capable of competing for specifically binding to influenzavirus H5N1 or a fragment thereof with the binding molecule of theinvention. In a further step it may be determined, if the screenedbinding molecules that are capable of competing for specifically bindingto influenza virus H5N1 or a fragment thereof have neutralizingactivity. A binding molecule that is capable of competing forspecifically binding to influenza virus H5N1 or a fragment thereof withthe binding molecule of the invention is another part of the invention.In the above-described screening method, “specifically binding to thesame epitope” also contemplates specific binding to substantially oressentially the same epitope as the epitope bound by the a bindingmolecule of the invention. The capacity to block, or compete with, thebinding of the binding molecules to influenza virus H5N1 typicallyindicates that a binding molecule to be screened binds to an epitope orbinding site on influenza virus H5N1 that structurally overlaps with thebinding site on influenza virus H5N1 that is immunospecificallyrecognized by the binding molecules. Alternatively, this can indicatethat a binding molecule to be screened binds to an epitope or bindingsite which is sufficiently proximal to the binding siteimmunospecifically recognized by the binding molecules to sterically orotherwise inhibit binding of the binding molecules to influenza virusH5N1.

In general, competitive inhibition is measured by means of an assay,wherein an antigen composition, i.e. a composition comprising influenzavirus H5N1 or fragments thereof, is admixed with reference bindingmolecules, i.e. the binding molecules, and binding molecules to bescreened. Usually, the binding molecules to be screened are present inexcess. Protocols based upon ELISAs and Western blotting are suitablefor use in such simple competition studies. By using species or isotypesecondary antibodies one will be able to detect only the bound referencebinding molecules, the binding of which will be reduced by the presenceof a binding molecule to be screened that recognizes substantially thesame epitope. In conducting a binding molecule competition study betweena reference binding molecule and any binding molecule to be screened(irrespective of species or isotype), one may first label the referencebinding molecule with a detectable label, such as, e.g., biotin, anenzymatic, a radioactive or other label to enable subsequentidentification. Binding molecules identified by these competition assays(“competitive binding molecules” or “cross-reactive binding molecules”)include, but are not limited to, antibodies, antibody fragments andother binding agents that bind to an epitope or binding site bound bythe reference binding molecule, i.e. a binding molecule hereof, as wellas antibodies, antibody fragments and other binding agents that bind toan epitope or binding site sufficiently proximal to an epitope bound bythe reference binding molecule for competitive binding between thebinding molecules to be screened and the reference binding molecule tooccur. Preferably, competitive binding molecules hereof will, whenpresent in excess, inhibit specific binding of a reference bindingmolecule to a selected target species by at least 10%, preferably by atleast 25%, more preferably by at least 50%, and most preferably by atleast 75%-90% or even greater. The identification of one or morecompetitive binding molecules that bind to about, substantially,essentially or at the same epitope as the binding molecules is astraightforward technical matter. As the identification of competitivebinding molecules is determined in comparison to a reference bindingmolecule, i.e. a binding molecule of the invention, it will beunderstood that actually determining the epitope to which the referencebinding molecule and the competitive binding molecule bind is not in anyway required in order to identify a competitive binding molecule thatbinds to the same or substantially the same epitope as the referencebinding molecule.

EXAMPLES

The following illustrative Examples are provided.

Example 1 Construction of scFv Phage Display Libraries Using RNAExtracted from Memory B Cells

Peripheral blood was collected from normal healthy donors byvenapuncture in EDTA anti-coagulation sample tubes. A blood sample (45ml) was diluted twice with PBS and 30 ml aliquots were underlayed with10 ml Ficoll-Hypaque (Pharmacia) and centrifuged at 900×g for 20 minutesat room temperature without breaks. The supernatant was removedcarefully to just above the white layer containing the lymphocytic andthrombocytic fraction. Next, this layer was carefully removed (˜10 ml),transferred to a fresh 50 ml tube and washed three times with 40 ml PBSand spun at 400×g for 10 minutes at room temperature to removethrombocytes. The obtained pellet containing lymphocytes was resuspendedin RPMI medium containing 2% FBS and the cell number was determined bycell counting. Approximately 1×10⁸ lymphocytes were stained forfluorescent cell sorting using CD24, CD27 and surface IgM as markers forthe isolation of IgM memory B cells. A Becton Dickinson Digital Vantageapparatus set in Yield Mode was used for physical memory B cell sortingand isolation. Lymphocytes were gated as the small compact populationfrom the FSC/SSC window. Memory B cells (CD24+/CD27+) were subsequentlyseparated from naive B cells (CD24+/CD27−) and memory T cells(CD24−/CD27+). In a next step, IgM memory B cells (IgM+) were separatedfrom switch memory B cells (IgM−) using IgM expression. In this step IgMmemory B cells were sorted in a separate sample tube. 1×10⁵ cells werecollected in DMEM/50% FBS and after completion of the sort they werecentrifuged at 400×g for 10 minutes and lysed in 500 μl TRIZOL total RNAextraction solution (Invitrogen). The RNA was extracted from the lysissolution using 200 μl chloroform and isopropanol precipitation asdetailed in the TRIZOL solution protocol. Next, 1 μl Pellet Paint(Novogen) was applied to enhance and visualize the pelleting process.The complete RNA preparation was dissolved in 23 μl DEPC treatedultrapure water (Invitrogen) and used for cDNA conversion withSuperScript III Reverse Transcriptase (Invitrogen). 1 μl Random Hexamers(500 ng/μl) (Promega) was added to the RNA sample and mixed and meltedat 65° C. for 5 minutes in a heated lid PCR machine. The sample wassnap-cooled on wet-ice and the following components were added: 8 μl5×RT buffer (250 mM Tris-HCl pH 8.3, 375 mM KCl, 15 mM MgCl₂), 2 μldNTPs (10 mM of each) (Invitrogen), 2 μl DTT (100 mM), 2 μl RNAseInhibitor (40 U/μl) (Promega), 2 μl SuperScript III (200 U/μl)(Invitrogen). The obtained mixture was first incubated for 5 minutes atroom temperature and then transferred to a heated lid PCR machine at 50°C. for one hour. The reaction was stopped by heating up to 75° C. for 15minutes The cDNA obtained was diluted to 200 μl with ultrapure water andstored at −20° C. until further use.

A two round PCR amplification approach was applied using the primer setsshown in Tables 1 and 2 to isolate the immunoglobulin VH and VL regionsfrom the respective donor repertoire. The PCR formulation foramplification used throughout the procedure was as follows: 5 μl cDNAtemplate, 32.75 μl ultrapure water, 2.5 μl of each primer (10 μM), 5 μl10×PCR buffer (200 mM Tris-HCl pH 8.4, 500 mM KCl), 1.5 μl MgCl₂ (50mM), 0.5 μl dNTPs (25 mM of each), 0.25 μl Taq polymerase (5 U/μl)(Invitrogen). First round amplification on the respective cDNA using theprimer sets mentioned in Table 1 yielded 7, 6 and 9 products of about650 base pairs for respectively VH, Vkappa and Vlambda regions. For IgMmemory B cell VH region amplification the OCM constant primer was usedin combination with OH1 to OH7. The thermal cycling program for firstround amplifications was: 2 minutes 96° C. (denaturation step), 30cycles of 30 sec 96° C./30 sec 55° C./60 sec 72° C., 10 minutes 72° C.final elongation and 4° C. refrigeration. The products were loaded onand isolated from a 1% agarose gel using gel-extraction columns (Qiagen)and eluted in 50 μl 1 mM Tris-HCl pH 8.0. Ten percent of first roundproducts (5 μl) was subjected to second round amplification using theprimers mentioned in Table 2. These primers were extended withrestriction sites enabling the directional cloning of the respective VLand VH regions into phage display vector PDV-006. The PCR program forsecond round amplifications was as follows: 2 minutes 96° C.(denaturation step), 30 cycles of 30 sec 96° C./30 sec 60° C./60 sec 72°C., 10 minutes 72° C. final elongation and 4° C. refrigeration. Thesecond round products (˜350 base pairs) were first pooled according tonatural occurrence of J segments found in immunoglobulin gene products,resulting in 7, 6 and 9 pools for respectively the VH, Vkappa andVlambda variable regions (see Tables 3 and 4). To obtain a normalizeddistribution of immunoglobulin sequences in the immune library the 6Vkappa and 9 Vlambda light chain pools were mixed according to thepercentages mentioned in Table 3. This single final VL pool (3 mg) wasdigested overnight with SalI and NotI restriction enzymes, loaded on andisolated from a 1.5% agarose gel (˜350 base pairs) using Qiagengel-extraction columns and ligated in SalI-NotI cut PDV-006 vector (5000base pairs) as follows: 10 μl PDV-006 vector (50 ng/μl), 7 μl VL insert(10 ng/μl), 5 μl 10× ligation buffer (NEB), 2.5 T4 DNA Ligase (400 U/μl)(NEB), 25.5 μl ultrapure water (vector to insert ratio was 1:2).Ligation was performed overnight in a water bath of 16° C. Next, thevolume was doubled with water, extracted with an equal volume ofphenol-chloroform-isoamylalcohol (75:24:1) (Invitrogen) followed bychloroform (Merck) extraction and precipitated with 1 μl Pellet Paint(Novogen), 10 μl sodium acetate (3 M pH 5.0) and 100 μl isopropanol for2 hours at −20° C. The obtained sample was subsequently centrifuged at20,000×g for 30 minutes at 4° C. The obtained precipitate was washedwith 70% ethanol and centrifuged for 10 minutes at 20,000×g at roomtemperature. Ethanol was removed by vacuum aspiration and the pellet wasair dried for several minutes and then dissolved in 50 μl buffercontaining 10 mM Tris-HCl, pH 8.0. 1 μl ligation mixture was used forthe transformation of 40 μl TG-1 electro-competent cells (Stratagene) ina chilled 0.1 cm electroporation cuvette (Biorad) using a Genepulser IIapparatus (Biorad) set at 1.7 kV, 200 Ohm, 25 μF (time constant ˜4.5msec). Directly after pulse, the bacteria were flushed from the cuvettewith 1000 μl SOC medium (Invitrogen) containing 5% (w/v) glucose (Sigma)at 37° C. and transferred to a 15 ml round bottom culture tube. Another500 μl SOC/glucose was used to flush residual bacteria from the cuvetteand was added to the culture tube. Bacteria were recovered by culturingfor exactly one hour at 37° C. in a shaker incubator at 220 rpm. Thetransformed bacteria were plated over large 240 mm square petri dishes(NUNC) containing 200 ml 2TY agar (16 g/l bacto-tryptone, 10 g/lbacto-yeast extract, 5 g/l NaCl, 15 g/l agar, pH 7.0) supplemented with50 μg/ml ampicillin and 5% (w/v) glucose (Sigma). A 1 to 1000 dilutionwas plated for counting purposes on 15 cm petri dishes containing thesame medium. This transformation procedure was repeated sequentiallytwenty times and the complete library was plated over a total of thirtylarge square petri dishes and grown overnight in a 37° C. culture stove.Typically, around 1×10⁷ cfu were obtained using the above protocol. Theintermediate VL light chain library was harvested from the plates bymildly scraping the bacteria into 10 ml 2TY medium per plate. The cellmass was determined by OD600 measurement and two times 500 OD ofbacteria was used for maxi plasmid DNA preparation using two P500maxiprep columns (Qiagen) according to manufacturer's instructions.

Analogous to the VL variable regions, the second round VH-JH productswere first mixed together to obtain the normal J segment usagedistribution (see Table 4), resulting in 7 VH subpools called PH1 toPH7. The pools were mixed to acquire a normalized sequence distributionusing the percentages depicted in Table 4, obtaining one VH fractionthat was digested with SfiI and XhoI restriction enzymes and ligated inSfiI-XhoI cut PDV-VL intermediate library obtained as described above.The ligation set-up, purification method, subsequent transformation ofTG1 and harvest of bacteria was exactly as described for the VLintermediate library (see above). The final library (approximately 5×10⁶cfu) was checked for insert frequency with a colony PCR using a primerset flanking the inserted VH-VL regions. More than 95% of the coloniesshowed a correct length insert (see Table 5). The colony PCR productswere used for subsequent DNA sequence analysis to check sequencevariation and to assess the percentage of colonies showing a completeORF. This was typically above 70% (see Table 5). The frequency ofmutations in the V genes was also analyzed. Out of 50 sequences, 47(94%) were not in germline configuration indicative of a maturationprocess and consistent with the memory phenotype of the B cells used asan RNA source for the library. Finally, the library was rescued andamplified by using CT helper phages (see WO 02/103012) and was used forphage antibody selection by panning methods as described below.

Example 2 Selection of Phages Carrying Single Chain Fv Fragments AgainstH5N1

Antibody fragments were selected using antibody phage display librariesconstructed essentially as described above and general phage displaytechnology and MABSTRACT® technology essentially as described in U.S.Pat. No. 6,265,150 and in WO 98/15833 (both of which are incorporated byreference herein). Furthermore, the methods and helper phages asdescribed in WO 02/103012 (which is incorporated by reference herein)were used in the invention.

Selection was performed against recombinant hemagglutinin (HA) subtypeH5 (A/Vietnam/1203/2004; see SEQ ID NO:110) produced using baculovirusvectors in insect cells (Protein Sciences, CT, USA). The amino acidsequence of HA from isolate A/Vietnam/1194/2004 (H5N1TV) and theconsensus amino acid sequence representing HAs from strains isolated inIndonesia and China (H5N1IC) are shown in SEQ ID NO:111 and SEQ IDNO:112, respectively. Selections were also performed against solublerecombinant HA (sHA) from H5N1. The sequence encoding for theextracellular (soluble) part of HA (sHA) from isolateA/Vietnam/1194/2004 (H5N1), representing the HAs identified in influenzastrains isolated in Thailand and Vietnam (sHA of H5N1TV, SEQ ID NO:113)in 2004 (clade 1) and a consensus sequence representing soluble parts ofHAs of H5N1 strains isolated in Indonesia and China (sHA of H5N1IC, SEQID NO:114) in 2003/2004 (clade 2) were cloned in an expression vectorcontaining a myc- and his-tag using standard DNA cloning techniques. HAsof H5N1TV and

H5N1IC differ at 9 amino acid positions, all located in the HA1 subunitof the molecules. DNA transfections were performed in PER.C6® cells fortransient and/or stable expression using standard techniques. sHA waspurified from culture supernatant using metal chelate affinitychromatography using H isTrap™ FF Columns (Amersham Biosciences)according to the manufacturer's instructions.

HA antigens (recombinant HA and sHA of H5N1TV) were diluted in PBS (5.0μg/ml), added to MaxiSorp™ Nunc-Immuno Tubes (Nunc) and incubatedovernight at 4° C. on a rotating wheel. The immunotubes were emptied andwashed three times in block buffer (2% non-fat dry milk (ELK) in PBS).Subsequently, the immunotubes were filled completely with block bufferand incubated for 1-2 hours at room temperature. In addition,immunotubes were coated overnight with anti-myc antibody and incubatedwith block buffer containing 5 μg/ml myc-tagged sHA of H5N1TV. Aliquotsof pooled phage display library (500-1000 μl, 0.5×10¹³-1×10¹³ cfu,amplified using CT helper phage (see WO 02/103012)) were blocked inblocking buffer supplemented with 10% non-heat inactivated fetal bovineserum and 2% mouse serum for 1-2 hours at room temperature. The blockedphage library was added to the immunotubes, incubated for 2 hours atroom temperature, and washed with wash buffer (0.05% (v/v) TWEEN®-20 inPBS) to remove unbound phages. Bound phages were eluted from therespective antigen by incubation with 1 ml of 100 mM triethylamine (TEA)for 10 minutes at room temperature. Subsequently, the eluted phages weremixed with 0.5 ml of 1 M Tris-HCl pH 7.5 to neutralize the pH. Thismixture was used to infect 5 ml of an XL1-Blue E. coli culture that hadbeen grown at 37° C. to an OD 600 nm of approximately 0.3. The phageswere allowed to infect the XL1-Blue bacteria for 30 minutes at 37° C.Then, the mixture was centrifuged for 10 minutes at 3000×g at roomtemperature and the bacterial pellet was resuspended in 0.5 ml 2-tryptonyeast extract (2TY) medium. The obtained bacterial suspension wasdivided over two 2TY agar plates supplemented with tetracycline,ampicillin and glucose. After incubation overnight of the plates at 37°C., the colonies were scraped from the plates and used to prepare anenriched phage library, essentially as described by De Kruif et al.(1995a) and WO 02/103012. Briefly, scraped bacteria were used toinoculate 2TY medium containing ampicillin, tetracycline and glucose andgrown at a temperature of 37° C. to an OD 600 nm of ˜0.3. CT helperphages were added and allowed to infect the bacteria after which themedium was changed to 2TY containing ampicillin, tetracycline andkanamycin. Incubation was continued overnight at 30° C. The next day,the bacteria were removed from the 2TY medium by centrifugation afterwhich the phages in the medium were precipitated using polyethyleneglycol (PEG) 6000/NaCl. Finally, the phages were dissolved in 2 ml ofPBS with 1% bovine serum albumin (BSA), filter-sterilized and used forthe next round of selection.

Alternatively, phage selections using full-length HA-expressing celllines were performed. To this end, expression vectors containing thecomplete coding sequence of full-length HA from isolateANietnam/1194/2004 (H5N1TV) and a consensus sequence representingfull-length HAs of H5N1 strains isolated in Indonesia and China (H5N1IC)were used to transfect PER.C6® cells. Flow cytometry analysis using ananti-HA antibody was performed in parallel with each phage selection toassure HA expression by H5N1TV- and H5N1IC-transfected PER.C6® cells(data not shown). 10×10⁶ untransfected PER.C6® cells were resuspended in0.5 ml pooled phage library and 0.5 ml PER.C6® culture mediumsupplemented with 4% ELK and incubated for 1 hour in an end-over-endrotor set at 5 rpm at 4° C. After 5 minutes centrifugation at 300×g at4° C. of the phage library/untransfected PER.C6® cell mixture, thesupernatant containing the phages was subtracted for a second time with10×10⁶ untransfected PER.C6® cells. The subtracted phage preparation wassubsequently mixed with 1×10⁶ HA-expressing PER.C6® cells and incubatedfor 2 hours in an end-over-end rotor set at 5 rpm at 4° C. Subsequently,the cells were spun for 2 minutes at 3000×g and the cell pellet wasresuspended in 1 ml PBS/0.05% TWEEN®-20 to wash away unbound phages. Thecells were centrifuged for 2 minutes at 3000×g and the washing wasrepeated for an additional 5 times. After each wash, the cells weretransferred to a new tube. Bound phages were eluted from the antigen byincubation with 1 ml of 100 mM TEA for 10 minutes in an end-over-endrotor set at 5 rpm at room temperature. The cells were spun for 2minutes at 3000×g and the eluted phages were transferred to a 50 ml tubecontaining 0.5 ml 1 M Tris-HCl, pH 7.5. Rescue and propagation of theeluted phages was performed as described above. Two rounds of selectionswere performed before isolation of individual single-chain phageantibodies. After the second round of selection, individual E. colicolonies were used to prepare monoclonal phage antibodies. Essentially,individual colonies were grown to log-phase in 96 well plate format andinfected with VCS-M13 helper phages after which phage antibodyproduction was allowed to proceed overnight. The supernatants containingphage antibodies were used directly in ELISA for binding to HA antigens.Alternatively, phage antibodies were PEG/NaCl-precipitated andfilter-sterilized for flow cytometry analysis.

Example 3 Validation of the HA Specific Single-Chain Phage Antibodies

Selected single-chain phage antibodies that were obtained in thescreening described above were validated in ELISA for specificity, i.e.binding to HA antigens. For this purpose, baculovirus expressedrecombinant HA (Protein Sciences, CT, USA) and purified sHAs of H5N1TVand H5N1IC were coated to Maxisorp™ ELISA plates. Anti-myc mA 9E10(Roche) was immobilized on Maxisorp™ ELISA plates as negative controlantigen. After coating, the plates were washed three times with PBScontaining 0.1% v/v TWEEN®-20 and blocked in PBS containing 3% BSA or 2%ELK for 1 hour at room temperature. The selected single-chain phageantibodies were incubated for 1 hour in an equal volume of PBScontaining 4% ELK to obtain blocked phage antibodies. The plates wereemptied, washed three times with PBS/0.1% TWEEN®-20 and the blockedsingle-chain phage antibodies were added to the wells. Incubation wasallowed to proceed for one hr, the plates were washed with PBS/0.1%TWEEN®-20 and bound phage antibodies were detected (using OD 492 nmmeasurement) using an anti-M13 antibody conjugated to peroxidase. As acontrol, the procedure was performed simultaneously without single-chainphage antibody and with a negative control single-chain phage antibody.From the selections on the different HA antigens with the IgM memory Bcell library, 92 unique single-chain phage antibodies specific foreither recombinant HA or sHA were obtained.

Alternatively, the reactivity of single chain antibodies that wereselected for binding to HA-expressing PER.C6® cells was tested usingflow cytometry analysis. PEG/NaCl precipitated phages were mixed with anequal volume of PBS/2% ELK blocked for 30 minutes on ice. The blockedphages were added to pelleted cells (untransfected PER.C6® andHA-expressing PER.C6® cells) and incubated for one hour on ice. Thecells were washed three times with PBS/1% BSA, followed by a 1 minutecentrifugation at 300×g, and binding of the single chain phageantibodies to the cells was visualized using a biotinylated anti-M13antibody (Fitzgerald) followed by streptavidin-phycoerythrin conjugate(Caltag). The selections using HA-expressing PER.C6® cells provided 24unique single-chain phage antibodies that were not identified duringselections using the different recombinant HA proteins.

Example 4 Characterization of the HA Specific scFvs

From the selected specific single-chain phage antibodies (scFv) clonesplasmid DNA was obtained and nucleotide and amino acid sequences weredetermined according to standard techniques. The nucleotide and aminoacid sequence and VH and VL gene identity (see Tomlinson I M et al.V-BASE Sequence Directory. Cambridge United Kingdom: MRC Centre forProtein Engineering (1997)) of the scFvs are depicted in Table 6. TheCDR regions of the HA-specific immunoglobulins are shown in Table 7.

Example 5 Construction of Fully Human Immunoglobulin Molecules (HumanMonoclonal Antibodies) from the Selected Single Chain Fvs

Heavy and light chain variable regions of the scFvs were cloned directlyby restriction digest for expression in the IgG expression vectorspIg-C911-HCgamma1 (see SEQ ID NO:141), pIG-C909-Ckappa (see SEQ IDNO:142), or pIg-C910-Clambda (see SEQ ID NO:143). Nucleotide sequencesfor all constructs were verified according to standard techniques knownto the skilled artisan. The resulting expression constructs encoding thehuman IgG1 heavy and light chains were transiently expressed incombination in 293T cells and supernatants containing human IgG1antibodies were obtained and produced using standard purificationprocedures. The human IgG1 antibodies were titrated in a concentrationrange of between 10 and 0.003 μg/ml against H5 (data not shown). ASARS-CoV specific antibody was included as a control antibody. The IgG1molecules showed the same pattern of reactivity as demonstrated for thesingle-chain phage antibodies.

The nucleotide and amino acid sequences of the heavy and light chain ofthe antibodies CR6141, CR6255, CR6257, CR6260, CR6261, CR6262, CR6268,CR6272, CR6296, CR6301, CR6307, CR6310, CR6314, CR6323, CR6325, CR6327,CR6328, CR6329, CR6331, CR6332, CR6334, CR6336, CR6339, CR6342, CR6343and CR6344 and their heavy and light chain variable regions are given inTable 8. Subsequently, binding of the anti-HA IgG to HA-expressingPER.C6® cells was investigated by flow cytometry. Flow cytometryanalysis for antibody binding to HA demonstrated that antibodies CR6255,CR6257, CR6260, CR6261, CR6262, CR6268, CR6307, CR6310, CR6314, CR6323,CR6325, CR6331 and CR6344 bound to HA (H5N1TV)-expressing PER.C6® cells(see Table 9). Antibodies CR6261 and CR6344 also displayed binding tountransfected control cells, but the obtained signals were approximately10-fold lower than the signals obtained for binding to HA(H5N1TV)-expressing PER.C6® cells (see Table 9). No binding of controlantibody CR3014 to HA-expressing cells and untransfected control cellswas observed.

Example 6 In Vitro Neutralization of H5N1 Influenza Virus by H5N1Specific IgGs (Virus Neutralization Assay)

In order to determine whether the selected IgGs were capable of blockingH5N1 infection, in vitro virus neutralization assays (VNA) wereperformed. The VNA were performed on MDCK cells (ATCC CCL-34). MDCKcells were cultured in MDCK cell culture medium (MEM medium supplementedwith antibiotics, 20 mM Hepes and 0.15% (w/v) sodium bicarbonate(complete MEM medium), supplemented with 10% (v/v) fetal bovine serum).The H5N1 reassortant strain NIBRG-14 which was used in the assay wasdiluted to a titer of 4×10³ TCID50/ml (50% tissue culture infective doseper ml), with the titer calculated according to the method of Spearmanand Karber. The IgG preparations (200 ng/ml) were serially 2-folddiluted (1:2-1:16) in complete MEM medium in duplicate wells. 25 μl ofthe respective IgG dilution was mixed with 25 μl of virus suspension(100 TCID50/25 n1) and incubated for one hour at 37° C. The suspensionwas then transferred in duplicate onto 96-well plates containingconfluent MDCK cultures in 50 μl complete MEM medium. Prior to use, MDCKcells were seeded at 3×10⁴ cells per well in MDCK cell culture medium,grown until cells had reached confluence, washed with 300-350 μl PBS, pH7.4 and finally 50 μl complete MEM medium was added to each well. Theinoculated cells were cultured for 3-4 days at 33° C. and observed dailyfor the development of cytopathic effect (CPE). CPE was compared to thepositive control (NIBRG-14-inoculated cells) and negative controls(mock-inoculated cells). The complete absence of CPE in an individualcell culture was defined as protection. Sheep anti-ANietnam/1194/04 H5N1influenza virus HA (04/214, NIBSC) was used as a positive control in theassay.

In addition, cultures were tested for the presence of virus usingimmunohisto-chemistry. To this end, the culture supernatant wasdiscarded and the cells were fixed with 40% (v/v) acetone and 60% (v/v)methanol for 15 minutes Fixed cells were blocked for 30 minutes at 37°C. in blocking buffer (200 mM NaCl, 0.2% (w/v) bovine serum albumin(BSA), 0.01% thimerosal, 0.2% (v/v) TWEEN®-20, 20 mM Tris-HCl, pH 7.45)supplemented with 2% (w/v) BSA and 5% (v/v) goat serum. Subsequently,cells were incubated for one hour at 37° C. with 50 μl mouseanti-influenza A monoclonal antibody blend (Chemicon) diluted 1:1000 inwashing buffer. After three washes with washing buffer, 50 μl ofbiotin-conjugated goat anti-mouse IgG (Jackson) diluted 1:1000 inwashing buffer was added and incubated for one hour at 37° C. Afterthree washes with washing buffer, 50 μl of streptavidin-peroxidaseconjugate (Calbiochem) diluted 1:3000 in washing buffer was added andincubated for 30 minutes at 37° C. After another three washes withwashing buffer, staining was visualized using AEC solution (0.12% (w/v)3-amino-9-ethylcarbazole, 30% (v/v) N—N-dimethylformamide and 70% (v/v)acetate buffer) containing 1 μl H₂O₂/1 ml AEC solution. After threewashes with washing buffer, staining was analyzed under a microscope.

The human anti-HA antibodies called CR6255, CR6257, CR6260, CR6261,CR6262, CR6268, CR6307, CR6310, CR6314, CR6323, CR6325, CR6331 andCR6344 were subjected to the above-described VNA. All antibodiesneutralized H5N1 reassortant strain NIBRG-14. The concentrations (inμg/ml) at which these antibodies protect MDCK cultures against CPE aregiven in Table 10. In wells where CPE was observed, the presence ofvirus was confirmed by immunohistochemical staining (data not shown).

In order to determine the neutralizing potency of the H5N1 specific IgGsmore accurately, the in vitro virus neutralization assays with NIBRG-14was repeated, but this time the IgG preparations were further diluted.The IgG preparations (200 μg/ml) were serially 2-fold diluted(1:1-1:512) in complete MEM medium and tested in quadruplicate wells inthe VNA as described above. The neutralizing potency of the humananti-H5N1 antibodies called CR6255, CR6257, CR6260, CR6261, CR6262,CR6268, CR6272, CR6307, CR6310, CR6314, CR6323, CR6325, CR6327, CR6328,CR6329, CR6331, CR6332, CR6334, CR6336, CR6339, CR6342, CR6343 andCR6344 was determined in the VNA. All antibodies, except CR6272 andCR6339, neutralized H5N1 reassortant strain NIBRG-14. The concentrations(in μg/ml, in the presence of 100 TCID50 of virus) at which theseantibodies protect MDCK cultures against CPE are indicated in Table 11.In wells where CPE was observed, the presence of virus was confirmed byimmunohistochemical staining (data not shown).

Example 7 Immunoblot Analysis of H5N1 Specific IgGs

To further investigate the specificity of the anti-HA antibodies,different recombinant hemagglutinin Influenza A antigens were subjectedto SDS-PAGE under reducing conditions followed by anti-HA immunoblotanalysis. The HA0 polypeptide is composed of the HA1 and HA2 subunit.Antibody CR5111, a H5N1-specific control antibody, recognized the HA1subunit and the intact (uncleaved) HA0 polypeptide of sHA of H5N1TV (seeFIG. 1, right part, lane 1). In addition, CR5111 recognized the HA1subunit of recombinant HA, subtype H5 (A/Vietnam/1203/2004 (H5N1); seeSEQ ID NO:110) produced using baculovirus vectors in insect cells(Protein Sciences, CT, USA) (FIG. 1, right part, lane 2). Uncleaved HA0polypeptide was not detected in this HA preparation. The difference insize between the HA subunits in lanes 1 and 2 might be explained by adifferent glycosylation of HA expressed in insect cells and PER.C6®cells. CR5111 did not recognize the HA1 and/or HA0 polypeptides ofrecombinant HA, subtype H1 (A/New Calcdonia/20/99 (H1N1)) (see FIG. 1,right part, lane 4) or of recombinant HA, subtype H3(A/Wyoming/3/2003(H₃N₂)) (FIG. 1, right part, lane 3).

Antibodies CR6307 and CR6323 recognized the HA2 subunits of sHA ofH5N1TV (see FIG. 1, left and middle part, lanes 1) and recombinant HA,subtype H5 (A/Vietnam/1203/2004 (H5N1) (see FIG. 1, left and middlepart, lanes 2). Interestingly, the HA2 subunit as present in the intact,uncleaved HA0 polypeptides was not recognized. Apparently, the epitoperecognized by CR6307 and CR6323 becomes accessible upon cleavage of HA0.In addition, antibodies CR6307 and CR6323 recognized the HA2 subunit ofrecombinant HA, subtype H1 (A/New Calcdonia/20/99 (H1N1)) (see FIG. 1,left and middle part, lanes 4), but not the HA2 subunit of recombinantHA, subtype H3 (A/Wyoming/3/2003(H₃N₂)) (see FIG. 1, left and middlepart, lanes 3), both recombinant HAs produced using baculovirus vectorsin insect cells (Protein Sciences, CT, USA). Because the binding site ofneutralizing antibodies CR6307 and CR6323 is conserved within influenzaA strains of the subtype H1 and H5, a molecule comprising the bindingsite could be considered as (part of a) vaccine capable of inducing abroadly cross-reactive anti-influenza antibody response.

Next to antibodies CR6307 and CR6323, the antibodies called CR6141,CR6296, and CR6301 were used in immunoblot analysis. Each of the threeantibodies was able to bind to the HA2 subunit of sHA of H5N1TV andrecombinant HA, subtype H5 (A/Vietnam/1203/2004; H5N1) (data not shown).

Example 8 FACS Analysis to Determine the Subunit Specificity of H5N1Specific IgGs

To evaluate the contribution of the HA1 subunit to binding of anti-H5N1antibodies, an assay was set up, wherein the HA1 subunit is releasedfrom HA-expressing PER.C6® cells. HA-expressing PER.C6® cells werewashed three times with PBS, pH 7.4, and incubated for 10 minutes inacidified PBS, pH 4.9. Subsequently, the cells were washed with PBS, pH7.4, and treated for 10 minutes with 10 μg/ml trypsin in PBS, pH 7.4, tocleave HA0 molecules into disulfide bond-linked HA1 and HA2 subunits.Finally, the cells were incubated for 20 minutes in 20 mM DTT in PBS, pH7.4, to release the HA1 subunit from the membrane-anchored HA2 subunit.Untreated and acid/trypsin/DTT-treated cells were washed twice with PBScontaining 1% w/v BSA. For flow cytometry analysis, 2.5×10⁵ cells wereused per staining. No staining of treated and untreated cells with thenegative control antibody CR3014 was observed. Antibody CR5111, whichbinds to the HA1 subunit in immunoblot analysis, stained the populationof untreated HA-expressing PER.C6® cells, while treated cells were notrecognized (data not shown). This provides further proof that CR5111recognizes the HA1 subunit. Treated and untreated cells were stained toa similar extent by the antibodies CR6307 and CR6323 (data not shown).Release of the HA1 subunit obviously did not influence binding of theseantibodies further substantiating the results from the immunoblotanalysis that the antibodies bind to the HA2 subunit. Antibodies CR6325and CR6331 bound to untreated cells, while binding to treated cells wasmarkedly reduced compared with binding to untreated cells (data notshown). This suggests that the affinity of the antibodies for theirepitope is significantly reduced by the conformational change inducedupon acid treatment or by the reduction of the disulfide bond that linksthe HA1 and HA2 subunit.

Example 9 Hemagglutinin Competition ELISA with Phage Antibodies and IgGs

To identify antibodies that bind to non-overlapping, non-competingepitopes, a hemagglutinin competition ELISA was performed. Nunc-Immuno™Maxisorp F96 plates were coated overnight at 4° C. with 0.5 μg/mlrecombinant HA, subtype H5 (A/Vietnam/1203/2004 (H5N1) (ProteinSciences) in PBS. Uncoated protein was washed away before the wells wereblocked with 300 μl PBS containing 2% w/v non-fat dry milk (blockingsolution) for 1 hour at room temperature. The blocking solution wasdiscarded and 50 μl of 10 μg/ml anti-H5N1 IgG in blocking solution wasincubated per well for 1 hour at room temperature. Subsequently, 50 μlof phage antibody in blocking solution in a concentration twice of theconcentration that resulted in 50% maximal binding (as determined in aprevious assay) was added per well and incubated for another hour atroom temperature. Wells were washed three times with PBS containing 0.1%v/v TWEEN®-20. Bound phage antibody was detected using aperoxidase-conjugated anti-M13 antibody. Wells were washed again asdescribed above and the ELISA was further developed by the addition of100 μl of OPD reagent (Sigma). The reaction was stopped by adding 50 μl1 M H₂SO₄ and then the OD at 492 nm was measured. Binding of phageantibody, in the presence of IgG, was expressed as percentage of bindingin the absence of IgG, which was set at 100%. Phage antibody SC05-111and corresponding IgG CR5111, which recognize an epitope in the HA1subunit of H5 hemagglutinin were included as controls.

The results indicate that the majority of the phage antibodies and IgGsbind to an overlapping epitope on recombinant HA, subtype H5(A/Vietnam/1203/2004 (H5N1)) (data not shown). Binding of SC05-111 phageantibody was blocked by CR5111 IgG, but not by any other IgG, suggestingthat the other IgGs recognize an epitope different from the bindingregion of CR5111. Five IgGs, CR6262, CR6272, CR6307, CR6339 and CR6343competed to a lower extent for binding than the rest of the IgGs (morethan 25% residual binding). For IgGs CR6262, CR6272, CR6339, and CR6343this can be explained by a lower affinity for HA. This is supported bythe observation that the phage antibodies corresponding to theseantibodies were competed away more efficiently by the other IgGs.Binding of phage antibody SC06-307 is blocked by IgG CR6307, but lessefficiently by the other IgGs. This suggests that SC06-307 and CR6307recognize a unique epitope which differs from the epitope recognized bythe other IgGs.

Example 10 Cross-Reactivity ELISA Using Anti-H5N1 IgGs

To test whether the epitope of the anti-H5N1 antibodies is conservedamong HAs other than subtype H5, a hemagglutinin cross-reactivity ELISAwas performed. Nunc-Immuno™ Maxisorp F96 plates (Nunc) were coatedovernight at 4° C. with 0.5 μg/ml recombinant HA, subtype H1 (A/NewCalcdonia/20/99 (H1N1)), subtype H3 (A/Wyoming/3/03 (H₃N₂)), subtype H5(A/Vietnam/1203/04 (H5N1)), subtype H7 (A/Netherlands/219/03 (H7N7)),and subtype H9 (A/Hong Kong/1073/99 (H9N2)) (Protein Sciences Corp.) inPBS. Additionally, BPL-inactivated virus preparations containing 0.5μg/ml of HA of A/New Calcdonia/20/99 (H1N1), and reassortant strainNIBRG-14 (A/Vietnam/1194/04 (H5N1)) were coated on the plates overnightin PBS. Wells were washed three times with PBS containing 0.1% v/vTWEEN®-20 to remove uncoated protein and subsequently blocked with 300μl PBS containing 2% w/v non-fat dry milk (blocking solution) for 1 hourat room temperature. The blocking solution was discarded and 100 μl perwell of 5 μg/ml anti-H5N1 antibodies in blocking solution was incubatedfor 1 hour at room temperature. Wells were washed three times with PBScontaining 0.1% v/v TWEEN®-20 and bound antibodies were detected using aperoxidase-conjugated mouse anti-human IgG antibody (Jackson). Thereaction was developed and measured as described supra. Table 12 showsthat all anti-H5N1 IgGs bound to recombinant HA, subtype H5(A/Vietnam/1203/2004 (H5N1)) and a BPL-inactivated virus preparation ofNIBRG-14, which contains the HA of strain ANietnam/1194/2004 (H5N1).Recombinant HAs of subtype H3 and H7 were not recognized by any of thetested anti-H5N1 IgGs. Interestingly, all anti-H5N1 IgGs, with theexception of CR5111 and CR6307, bound to recombinant HA of subtypes H1and H9, and a BPL-inactivated virus preparation of strain A/NewCalcdonia/20/99 (H1N1). This indicates that the epitope of the majorityof the anti-H5N1 IgGs is conserved among HA molecules of differentsubtypes.

Example 11 Epitope Mapping of Anti-H5N1 IgGs

Okuno et al. (1993) and Smirnov et al. (1999) demonstrated the existenceof a common epitope shared by the HAs of the influenza A virus subtypesH1, H2, and H5 and neutralization of these subtypes by the murinemonoclonal antibody C179 directed against this epitope. Thisconformational epitope is composed of two different sites which arelocated in the HA1 and HA2 subunit. Both sites are located in closeproximity in the middle of the stem region of the HA molecule. In orderto evaluate whether the anti-HA antibodies described supra recognizedthis epitope, HA molecules containing amino acid substitutions in thisregion were made.

The epitope recognized by antibody C179 (Takara Bio Inc.) has beenattributed to regions encompassing residues 318-322 of the HA1 subunitand residues 47-58 of the HA2 subunit (amino acid numbering as describedby Okuno et al. 1999). Escape viruses containing HAs that carry a Thr toLys substitution at position 318 in the HA1 subunit or a Val to Glusubstitution at position 52 in the HA2 subunit were no longer recognizedand neutralized by C179. These mutations (position 318 Thr to Lys,mutant I; position 52 Val to Glu, mutant II), a Leu to Met substitutionat position 320 in the HA1 subunit (mutant III, Met320 is present inregion 318-322 of HA1 of subtype H3 and H7), a Ser to Arg substitutionat position 54 in the HA2 subunit (mutant IV, Arg54 is present in region47-58 of HA2 of subtype H3 and H7) and an Asp to Asn substitution atposition 57 in the HA2 subunit (mutant V, Asn57 is present in region47-58 of HA2 of strain A/Hong Kong/156/97 (H5N1)) were introduced infull-length HA from isolate ANietnam/1194/2004 (H5N1TV) and transfectedin PER.C6® cells. Binding of C179 and the human anti-HA antibodies toPER.C6® cells expressing these mutants was evaluated by FACS analysis asdescribed supra. Antibody CR5111, which is directed against an epitopein the HA1 subunit, and a polyclonal anti-H5 sheep serum confirmed theexpression of H5N1TV and HA mutants by transfected PER.C6® cells (datanot shown). No staining with the negative control antibody CR3014 or inthe absence of antibody was observed (data not shown). As expected,antibody C179 did not recognize mutants I and II, which carried the sameamino acid substitutions as the HAs in the C179 escape viruses (seeOkuno et al., 1993). Furthermore, C179 did not bind to mutant IV,whereas binding of C179 to mutants III and V was unaffected. A similarpattern of reactivity was observed for antibody CR6342, which suggeststhat antibody C179 and CR6342 recognize a similar epitope. AntibodiesCR6261, CR6325 and CR6329 recognized all mutants, with the exception ofmutant II. This suggests that the epitope of antibodies CR6261, CR6325and CR6329 is different from that of C179. Since substitutions in theHA1 subunit did not abrogate binding of these antibodies, their epitopeis most likely located in the HA2 subunit. Antibodies CR6307 and CR6323recognized all mutants, which suggests that also the epitope of theseantibodies is different from that of C179. A summary of the sequence ofthe mutants and binding of the antibodies is given in Table 13. Inconclusion, the results indicate that antibodies CR6261, CR6325, CR6329,CR6307 and CR6323 are ideal candidates to bind to and neutralizeinfluenza viruses that have mutations in the epitope recognized by themurine monoclonal antibody C179 and as a consequence thereof are nolonger neutralized by this antibody.

Example 12 Prophylactic Activity of Human IgG Monoclonal AntibodiesAgainst Lethal H5N1 Challenge In Vivo

A lethal dose of influenza H5N1 strain A/HongKong/156/97 wasadministered to mice in order to study the prophylactic effect of humanmonoclonal IgG antibodies CR6261, CR6323 and CR6325. One day prior toinfection, 8 groups of 10 mice each were injected intraperitoneally withdifferent doses of antibody. As a negative control, one group of micewas injected with a non-relevant control antibody (CR3014). Clinicalsigns, weight loss and mortality were monitored until 21 days afterinfection. This study was conducted to assess the prophylactic effect ofthe monoclonal human anti-H5N1 IgG antibodies in vivo.

The H5N1 strain was originally obtained from a 3-year-old childsuffering from respiratory disease. The virus was passaged two times onMDCK cells. The batch [Titre 8.1 log TCID₅₀/ml] used to infect mice waspropagated once in embryonated eggs.

80 female 7-week-old Balb/c mice were divided in the 8 groups of 10 miceeach with the following injections prior to challenge with the H5N1virus:

-   -   1. 15 mg/kg CR6261.    -   2. 5 mg/kg CR6261.    -   3. 2 mg/kg CR6261.    -   4. 0.7 mg/kg CR6261.    -   5. 15 mg/kg CR6323.    -   6. 15 mg/kg CR6325    -   7. 500 μl rabbit anti-H5N3 immune serum (100× diluted).    -   8. 15 mg/kg CR3014.

All animals were acclimatized and maintained for a period of 6 daysprior to the start of the study. One day prior to infection with H5N1virus, 500 μl of antibody was administered by intraperitoneal injection.The animals were inoculated intranasally on day 0 with 25 LD₅₀ of virus(approximately 50 μl), and followed for 21 days. The actual dose of thevirus administered was estimated by titrating a few replicate samplesfrom the inoculum remaining after inoculation of the animals wascompleted. Virus titers (TCID50/mL) of the inoculum were determined onMDCK cells. The results showed that no inactivation of virus hadunintentionally occurred during preparation or administration of theinoculum. Group 8 acted as negative control. The animals in this groupwere injected with an irrelevant monoclonal antibody (CR3014) on day 0.Group 7 was supposed to act as positive control. The mice in this groupwere injected with a rabbit polyclonal serum antibody raised againstH5N3 influenza virus.

Clinical signs and weights were assessed daily from day −1 until 21 daysafter virus inoculation. Clinical signs were scored with a scoringsystem (0=no clinical signs; 1=rough coat; 2=rough coat, less reactive,passive during handling; 3=rough coat, rolled up, labored breathing,passive during handling; 4=rough coat, rolled up, labored breathing,does not roll back on stomach when laid down on its back) and recorded.Surviving animals were euthanized and bled on day 21. For analysis ofserum IgG antibody levels blood samples were collected from each mouseon day 0. Sera were prepared according to standard procedure. Togenerate post-infection sera, blood was collected from surviving animalson day 21. Sera were stored at −20° C.±2° C. until assayed for thepresence of virus specific antibodies. Sera were tested in duplicateusing 4 HAU of the H5N1 HK/97 substrate. Titers were expressed as thereciprocal of the highest serum dilution showing HI, starting at adilution of 1/10.

All mice were active and appeared healthy without showing signs ofdisease during the acclimatization period. The average clinical score(total clinical score divided by number of surviving animals in eachgroup) was calculated per day and the results are indicated in FIG. 2(average clinical score per group), Table 14 (clinical scores) and Table15 (respiratory distress). Onset of negative clinical signs was observedat day 3 after infection in the group that was inoculated with thenegative control Ab CR3014 (group 8). Respiratory distress was firstrecorded on day 6, and lasted for 1 to 4 days. No clinical signs wereobserved in mice that were treated with 15 mg/kg of CR6325 (group 6). Ingroup 5 (CR6323), one mouse showed mild clinical signs (score 1) fromday 5 to 8 and died the next day. In group 1, one mice died on day 13without having shown any previous clinical signs. Higher Ab doses ofCR6261 correlated with later onset and lower clinical scores on average.Respiratory distress was noticed in the lowest dose group (0.7 mg/kg),but not in the higher dose groups (2, 5 and 15 mg/kg). All mice showedimprovement in their clinical condition between day 9 and 13 (group 2, 3and 5) or from day 17 on (group 4). Mice that were injected with therabbit polyclonal antibody (group 7) developed severe illness within 3days and then died, demonstrating that the rabbit antibody did notprotect against infection in vivo. Two animals (one in group 4 and onein group 8) were euthanized on day 10 and removed from the study,because the animals were considered to be severely ill (score 4).

The animals infected with H5N1 showed varying degrees of weight loss(FIG. 3). Proportional weight loss and the moment of onset were relatedto antibody dose. In the groups treated with the highest dose ofantibodies weight steadily increased over time consistent withage-related weight gain. The group of animals inoculated with the lowestdose of CR6261 lost weight more quickly than the groups of animalsreceiving higher doses of antibody. The total amount of weight loss wasgreater in the lower dose groups, with average weight loss of about 15and 40% of starting weight in the groups inoculated with 2 and 0.7 mg/kgCR6261, respectively. In the lower dose groups average body weightappeared to increase again at the same time as animals showed someclinical improvement.

FIG. 4 shows the number of mice surviving per group on each day. A cleardose-response relationship between the amount of antibody administeredand average survival time was present. FIG. 5 shows the mortality in adose-responsive manner. The first mice died 7 days after inoculation inthe lowest dose group (0.7 mg/kg) and in the group of mice that receivedthe negative control: CR3014. Less than 50% of the animals wereprotected against death when 0.7 mg/kg of CR6261 antibody wasadministered. However, 9 to 10 mice survived when the highest dose ofantibody was administered. No mice of the negative control group(CR3014) survived, showing that indeed a 100% lethal challenge dose wasused in this study.

To assess whether IgG antibodies are able to render complete protectionagainst infection, an HI assay is performed with sera collected at day21 from mice that had received 15 mg/kg of CR6261 (group 1). This datashould indicate that the mice experienced an H5N1 infection albeitwithout clinical manifestation.

These results show that at least three human anti-H5N1 antibodies,identified and developed as disclosed herein (CR6261, CR6323 and CR6325)are each separately able to provide protection against a lethal dose ofinfluenza H5N1 in vivo. A clear dose-response relationship between theamount of CR6261 antibody administered and average survival time wasobserved. The results show that each monoclonal anti-H5N1 IgG antibodytested was able to prevent clinical manifestation of H5N1 infection inmice when administered one day prior to infection at a dose of 15 mg/kg.

Example 13 Protective and Therapeutic Effects of Human MonoclonalAnti-H5N1 Antibodies Administered after an Infection with a Lethal Doseof Influenza H5N1 Virus In Vivo

A study was performed to test the therapeutic effect of the monoclonalantibodies as disclosed herein, exemplified by CR6261, in apost-infection model, against a lethal H5N1 A/HK/97 influenza viruschallenge in vivo. The virus batch and the type, and age of mice werethe same as used in example 12. As a negative control one group of micewas injected with a non-relevant control antibody (CR2006). Clinicalsigns, weight loss and mortality were monitored until 21 days afterinfection.

58 female 7-week-old Balb/c mice were divided in 5 groups that receivedthe antibody at different stages after infection, as follows:

-   -   1. 10 mice; 15 mg/kg CR6261 at 4 hours post-infection    -   2. 14 mice; 15 mg/kg CR6261 at 1 day post-infection    -   3. 10 mice; 15 mg/kg CR6261 at 2 days post-infection    -   4. 10 mice; 15 mg/kg CR6261 at 3 days post-infection    -   5. 14 mice; 15 mg/kg CR2006 at 1 day post-infection

All animals were acclimatized and maintained for a period of 6 daysprior to the start of the study. The animals were inoculatedintranasally on day 0 with 25 LD₅₀ of H5N1 influenza virus(approximately 50 μl), and monitored for 21 days. The actual dose of thevirus administered was estimated by titrating a few replicate samplesfrom the inoculum remaining after inoculation of the animals wascompleted. Virus titers (TCID50/mL) of the inoculum were determined onMDCK cells. The results showed that no inactivation of virus hadunintentionally occurred during preparation or administration of theinoculum. At the specified time points after inoculation, 500 μl ofantibody was administered by intraperitoneal injection. Group 5 acted asnegative control. The animals in this group were injected with anirrelevant monoclonal antibody (CR2006) day 1 post-infection.

Clinical signs and weights were assessed each day from day −1 until day21. Clinical signs were scored as described in example 12, with ascoring range from 0 to 4. Surviving animals were euthanized and bled onday 21. For assessment of pathological changes, 4 animals of group 2 and5 were killed on day 6 after challenge. These animals were alreadypre-selected on day 0, and set apart from the others. For that reason,groups 2 and 5 started with 14 animals, with 10 mice remaining after theselection. Clinical signs and weights were assessed daily from day −1until 21 days after virus inoculation.

All mice were active and appeared healthy without showing signs ofdisease during the acclimatization period. The average clinical score(total clinical score divided by number of surviving animals in eachgroup) was calculated per day and the results are indicated in FIG. 6(average clinical score per group), Table 16 (clinical scores) and Table17 (respiratory distress). Clearly, all groups contained mice thatshowed clinical signs already at day 1 after infection. Depending on thetime at which the antibody was administered, the clinical signsdiminished and in all groups clinical signs were absent again at day 15.In the control group 5, all animals suffered from severe clinical signsand all animals had died, or were euthanized because they reached level4 in the clinical scores, at day 9. This shows that again a lethal doseof influenza virus was administered to the animals. In Group 1, whereinthe animals already received the antibody 4 hours after infection, someanimals did not develop clinical signs, whereas others did. The numberof animals that did exhibit clinical signs that could be scored areprovided in Table 16. Since influenza virus can have a dramatic effecton the respiratory organs, also the respiratory distress was measuredand here provided in Table 17. FIG. 7 and Table 18 show the number ofsurviving animals and the mortality rate respectively for all groups.For unknown reasons, one animal in group 1 that received the antibody 4hours after infection died at day 10. All remaining animals in thegroups that received the antibody after the influenza infection survivedand were healthy at day 21. This is clearly shown in the body weightdata that was obtained from all mice. FIG. 8 shows the mean body weightin each group of mice during the 21 days of the study. Although the bodyweight of all mice decreased upon infection, the body weight did returnto normal levels after administration of the antibody. Clearly, thereturn to normal body weight levels depended on the timing of theantibody treatment, where animals that were treated 4 hours afterexposure, recovered rapidly and reached normal levels at day 7, theanimals that were treated 3 days after infection, reached their normalbody weight at day 17. All animals reached a similar and healthy bodyweight at the end of the study, at day 21. Clearly, all animals thatreceived the negative control antibody did not regain body weight andmeasurements stopped at death at day 9.

These results show that a post-infection treatment with a monoclonalantibody directed against H5N1 influenza virus, as disclosed herein andexemplified by antibody CR6261, can rescue mammalian subjects, as showedherein in mice, after challenge with a lethal dose of H5N1 influenzavirus. Even at a late stage, 3 days post-infection, the antibody is ableto reverse the clinical symptoms to a level in which no clinical signscould be monitored anymore. Strikingly, at day 21 post-infection, allantibody-treated animals reached normal body weight levels and did notshow any remaining clinical signs.

Example 14 In Vivo Cross Protection Against Lethal Challenge byHeterologous Influenza Subtypes Using Monoclonal Antibodies DirectedAgainst HA of H5N1

As disclosed supra (example 11), some of the antibodies hereofapparently recognize a single epitope in the HA2 domain of the H5hemagglutinin protein. In example 7 it was shown that certain bindingmolecules hereof could interact with the HA2 epitope in anon-conformational manner, in other words, the form in which thehemagglutinin protein is folded does not seem to hinder the neutralizingactivity and the protective effects of these antibodies as disclosedherein.

The sequences of the influenza hemagglutinin protein parts that are notprone to antigenic drift (=the change of immunodominant regions withinthe HA1 region of the protein, resulting in the need for yearly updatedinfluenza vaccines) are known in the art. The epitope in HA2 that isrecognized by CR6261, CR6325 and CR6329 is contained in the amino acidsequence GVTNKVNSIIDK (SEQ ID NO:368, see Table 13) and is also presentin the hemagglutinin proteins of H1 and H9 subtypes, see Table 22. Itwas shown in example 10 that interaction of the binding molecules to theepitope was not limited to HA from H5, but that also HA from H1 and H9were recognized (see Table 12). Hence, the binding molecules recognizean epitope that is present in multiple HA proteins from differentinfluenza serotypes.

To study the cross-applicability of these antibodies for therapy invivo, an experiment was performed in mice that was in line with thedosing schedule of the experiment described in example 13. In thepresent experiment, a very high, lethal dose of another influenza virusrelated to the 1918 pandemic outbreak and now circulating seasonally inhumans, namely H1N1, was administered in mice. Subsequently, the micewere treated with an antibody of the invention, exemplified by CR6261,and clinical signs, respiratory distress and body weight were monitoredfor 3 weeks after infection. It turned out that these binding moleculesthat could rescue mice when infected with H5N1, were also able to rescuemammalian subjects when infected with H1N1, as outlined below.

A study was performed to test the therapeutic effect of the monoclonalantibodies, exemplified by CR6261, in a post-infection model against alethal H1N1 A/WSN/33 influenza virus challenge in vivo. The virus batchwas obtained from ATCC (VR-219) and was once propagated in embryonatedeggs. The titer was 8.5 log TCID₅₀/ml. As a negative control one groupof mice was injected with an irrelevant monoclonal antibody (IgG1, λnamed “CR57,” isotype matched negative control antibody). Clinicalsigns, weight loss and mortality were monitored until 21 days afterinfection.

50 female 6 to 8-week-old Balb/c mice were divided in 5 groups thatreceived the antibody at different stages related to the infection, asfollows:

-   -   1. 10 mice; 15 mg/kg CR6261 at 1 day prior to infection    -   2. 10 mice; 15 mg/kg CR6261 at 1 day post-infection    -   3. 10 mice; 15 mg/kg CR6261 at 2 days post-infection    -   4. 10 mice; 15 mg/kg CR6261 at 3 days post-infection    -   5. 10 mice; 15 mg/kg neg. contr. CR57 at 1 day-post infection

All animals were acclimatized and maintained for a period of at least 4days prior to the start of the study. The animals were inoculatedintranasally on day 0 with a lethal dose of H1N1 virus (6.6 log TCID₅₀;equivalent of 25× the LD₅₀) in approximately 50 μl, and monitored. Atthe specified time points before/after inoculation, 500 μl of antibodywas administered by intraperitoneal injection. General health of themice was monitored throughout the study. Clinical signs and weights wereassessed each day from day −1 until day 21. Clinical signs were scoredwith the scoring rates as disclosed in example 12 and 13, ranging from 0to 4. Surviving animals were euthanized and bled on day 21.

The mortality rate for each group is provided in Table 19, showing thenumber of living mice in each study group throughout the study. Two micedied shortly after the inoculation event (at day 1, one in group 2 andone in group 3) and were excluded from the analysis as pre-defined inthe study plan. All mice in the control group 5 were dead at day 9 likein the previous study with H5N1 (see Table 18). The percentage ofanimals surviving this lethal challenge dose of H1N1 is also plotted inFIG. 9. The number of mice showing relevant clinical signs in each groupis provided in Table 20. No clinical signs were observed in Group 1,whereas in Group 2, 3 and 4, some mice displayed clinical signs thatdisappeared completely after day 14 upon inoculation. All animals inGroup 5 started showing clinical signs at day 2. No mice in this grouprecovered. The number of mice showing respiratory distress in category 2or 3 is given in Table 21. No clinical distress was observed anymoreafter day 13 in Groups 1-4, whereas all remaining mice in control Group5 did suffer from severe respiratory distress.

FIG. 10 shows the average body weight of the mice in each study group.Clearly, no measurements are provided for the mice in Group 5 after day8. As can be seen from this figure, all mice that received the anti-H5N1antibody and that recovered from the clinical signs did get to theirexpected body weight level at day 21.

The antibodies could protect these mice when administered beforeinfection or after infection. Notably, the infection dose was ratherhigh: 25× the LD₅₀ dose, indicating that the antibodies provide a verystrong protection against the virus even when present at high titers inthe lung. This is clinically relevant as highly pathogenic viruses likeH5N1 replicate to high titer after infection and these high viral loadshave been linked to the frequently severe outcome in infected humans.Moreover, all protected mice recovered completely from this lethalinfection over time. It is concluded that the anti-H5N1 antibodieshereof (such as CR6261, CR6325, and CR6329) that bind to (single)epitopes in the HA2 region, a region that is not prone to antigenicdrift, provide cross-protection in vivo against multiple influenzaserotypes, circumventing the need for antibodies against the highlymutation-sensitive HA1 region. It is to be understood that the bindingmolecules that are not limited by epitopes that are only present in HAfrom an H5 influenza serotype, can also be used in the prophylactic-, ortherapeutic treatment of all influenza serotypes that contain the sameepitope in the stable region of HA2, such as H1N1, and influenza virusescomprising H2, H6 and H9 hemagglutinin proteins.

Example 15 Affinity Studies

Affinity studies were performed using surface plasmon resonance analysiswith a BIAcore3000 analytical system at 25° C. and 37° C., using HBS-EP(Biacore AB, Sweden) as running buffer at a flow rate of 75 μl/minute.IgGs were immobilized on a research grade CMS 4-flow channel (Fc) sensorchip (Biacore AB, Sweden) using amine coupling. A varying amount of HAfrom an H5N1 virus (ANietnam/1203/2004) was injected to analyze thebinding interaction between the HA protein and the immobilized IgGs.Regeneration using 20 mM NaOH was performed at the end of eachmeasurement to remove bound HA, while leaving the immobilized IgG on thechip.

Affinity constants were determined for CR6261, CR6323 and CR6325antibodies. Five concentrations in 4-fold dilutions of HA were injected(100 μl per injection), followed by a dissociation phase of 3600 sec,and regeneration using 10 μl 20 mM NaOH. The resulting data were fittedusing a 1:1 (Langmuir) model. However, an accurate dissociation constant(KD) could not be calculated. This was due to extremely low dissociationrates at 25° C. (even with an extended measurement) leading tounacceptable error in the calculation. When the experiments wererepeated at 37° C. discernible dissociation did occur but still notsufficiently enough to accurately measure the KD. Experiments areperformed to establish a definitive KD for the antibodies. These areestimated to be at least in the single digit nM range and most likely inthe pM range of affinity. These experiments show that the bindingmolecules have a very high affinity for their epitope present in the HAprotein of influenza virus.

TABLE 1 First round Vkappa, Vlambda and VH amplifications Primer namePrimer nucleotide sequence SEQ ID NO: OK1 (HuVK1B)GAC ATC CAG WTG ACC CAG TCT CC 144 OK2 (HuVK2)GAT GTT GTG ATG ACT CAG TCT CC 145 OK3 (HuVK2B2)GAT ATT GTG ATG ACC CAG ACT CC 146 OK4 (HuVK3B)GAA ATT GTG WTG ACR CAG TCT CC 147 OK5 (HuVK5)GAA ACG ACA CTC ACG CAG TCT CC 148 OK6 (HuVK6)GAA ATT GTG CTG ACT CAG TCT CC 149 OCK (HuCK)ACA CTC TCC CCT GTT GAA GCT CTT 150 OL1 (HuVL1A)*CAG TCT GTG CTG ACT CAG CCA CC 151 OL1 (HuVL1B)*CAG TCT GTG YTG ACG CAG CCG CC 152 OL1 (HuVL1C)*CAG TCT GTC GTG ACG CAG CCG CC 153 OL2 (HuVL2B)CAG TCT GCC CTG ACT CAG CC 154 OL3 (HuVL3A)TCC TAT GWG CTG ACT CAG CCA CC 155 OL4 (HuVL3B)TCT TCT GAG CTG ACT CAG GAC CC 156 OL5 (HuVL4B)CAG CYT GTG CTG ACT CAA TC 157 OL6 (HuVL5)CAG GCT GTG CTG ACT CAG CCG TC 158 OL7 (HuVL6)AAT TTT ATG CTG ACT CAG CCC CA 159 OL8 (HuVL7/8)CAG RCT GTG GTG ACY CAG GAG CC 160 OL9 (HuVL9)#CWG CCT GTG CTG ACT CAG CCM CC 161 OL9 (HuVL10)# CAG GCA GGG CTG ACT CAG162 OCL (HuCL2)X TGA ACA TTC TGT AGG GGC CAC TG 163 OCL (HuCL7)XAGA GCA TTC TGC AGG GGC CAC TG 164 OH1(HuVH1B7A)+CAG RTG CAG CTG GTG CAR TCT GG 165 OH1 (HuVH1C)+SAG GTC CAG CTG GTR CAG TCT GG 166 OH2 (HuVH2B)CAG RTC ACC TTG AAG GAG TCT GG 167 OH3 (HuVH3A) GAG GTG CAG CTG GTG GAG168 OH4 (HuVH3C) GAG GTG CAG CTG GTG GAG WCY GG 169 OH5 (HuVH4B)CAG GTG CAG CTA CAG CAG TGG GG 170 OH6 (HuVH4C)CAG STG CAG CTG CAG GAG TCS GG 171 OH7 (HuVH6A)CAG GTA CAG CTG CAG CAG TCA GG 172 OCM (HuCIgM)TGG AAG AGG CAC GTT CTT TTC TTT 173 * Mix in 1:1:1 ratio # Mix in 1:1ratio X Mix in 1:1 ratio + Mix in 1:1 ratio

TABLE 2 Second round Vkappa, Vlambda and VH amplifications Primer namePrimer nucleotide sequence SEQ ID NO: OK1S (HuVK1B-SAL)TGA GCA CAC AGG TCG ACG GAC ATC CAG 174 WTG ACC CAG TCT CCOK2S (HuVK2-SAL) TGA GCA CAC AGG TCG ACG GAT GTT GTG ATG 175ACT CAG TCT CC OK3S (HuVK2B2-SAL)TGA GCA CAC AGG TCG ACG GAT ATT GTG ATG 176 ACC CAG ACT CCOK4S (HuVK3B-SAL) TGA GCA CAC AGG TCG ACG GAA ATT GTG WTG 177ACR CAG TCT CC OK5S (HuVK5-SAL) TGA GCA CAC AGG TCG ACG GAA ACG ACA CTC178 ACG CAG TCT CC OK6S (HuVK6-SAL)TGA GCA CAC AGG TCG ACG GAA ATT GTG CTG 179 ACT CAG TCT CCOJK1 (HuJK1-NOT) GAG TCA TTC TCG ACT TGC GGC CGC ACG TTT 180GAT TTC CAC CTT GGT CCC OJK2 (HuJK2-NOT)GAG TCA TTC TCG ACT TGC GGC CGC ACG TTT 181 GAT CTC CAG CTT GGT CCCOJK3 (HuJK3-NOT) GAG TCA TTC TCG ACT TGC GGC CGC ACG TTT 182GAT ATC CAC TTT GGT CCC OJK4 (HuJK4-NOT)GAG TCA TTC TCG ACT TGC GGC CGC ACG TTT 183 GAT CTC CAC CTT GGT CCCOJK5 (HuJK5-NOT) GAG TCA TTC TCG ACT TGC GGC CGC ACG TTT 184AAT CTC CAG TCG TGT CCC OL1S (HuVL1A-SAL)*TGA GCA CAC AGG TCG ACG CAG TCT GTG CTG 185 ACT CAG CCA CCOL1S (HuVL1B-SAL)* TGA GCA CAC AGG TCG ACG CAG TCT GTG YTG 186ACG CAG CCG CC OL1S (HuVL1C-SAL)*TGA GCA CAC AGG TCG ACG CAG TCT GTC GTG 187 ACG CAG CCG CCOL2S (HuVL2B-SAL) TGA GCA CAC AGG TCG ACG CAG TCT GCC CTG 188 ACT CAG CCOL3S (HuVL3A-SAL) TGA GCA CAC AGG TCG ACG TCC TAT GWG CTG 189ACT CAG CCA CC OL4S (HuVL3B-SAL) TGA GCA CAC AGG TCG ACG TCT TCT GAG CTG190 ACT CAG GAC CC OL5S (HuVL4B-SAL)TGA GCA CAC AGG TCG ACG CAG CYT GTG CTG 191 ACT CAA TC OL6S (HuVL5-SAL)TGA GCA CAC AGG TCG ACG CAG GCT GTG CTG 192 ACT CAG CCG TCOL7S (HuVL6-SAL) TGA GCA CAC AGG TCG ACG AAT TTT ATG CTG 193ACT CAG CCC CA OL8S (HuVL7/8-SAL)TGA GCA CAC AGG TCG ACG CAG RCT GTG GTG 194 ACY CAG GAG CCOL9S (HuVL9-SAL)# TGA GCA CAC AGG TCG ACG CWG CCT GTG CTG 195ACT CAG CCM CC OL9S (HuVL10-SAL)#TGA GCA CAC AGG TCG ACG CAG GCA GGG CTG 196 ACT CAG OJL1 (HuJL1-NOT)GAG TCA TTC TCG ACT TGC GGC CGC ACC TAG 197 GAC GGT GAC CTT GGT CCCOJL2 (HuTh2/3-NOT) GAG TCA TTC TCG ACT TGC GGC CGC ACC TAG 198GAC GGT CAG CTT GGT CCC OJL3 (HuJL7-NOT)GAG TCA TTC TCG ACT TGC GGC CGC ACC GAG 199 GAC GGT CAG CTG GGT GCCOH1S (HuVH1B-SFI)+ GTC CTC GCA ACT GCG GCC CAG CCG GCC ATG 200GCC CAG RTG CAG CTG GTG CAR TCT GG OH1S (HuVH1C-SFI)+GTC CTC GCA ACT GCG GCC CAG CCG GCC ATG 201GCC SAG GTC CAG CTG GTR CAG TCT GG OH2S (HuVH2B-SFI)GTC CTC GCA ACT GCG GCC CAG CCG GCC ATG 202GCC CAG RTC ACC TTG AAG GAG TCT GG OH3S (HuVH3A-SFI)GTC CTC GCA ACT GCG GCC CAG CCG GCC ATG 203 GCC GAG GTG CAG CTG GTG GAGOH4S (HuVH3C-SFI) GTC CTC GCA ACT GCG GCC CAG CCG GCC ATG 204GCC GAG GTG CAG CTG GTG GAG WCY GG OH5S (HuVH4B-SFI)GTC CTC GCA ACT GCG GCC CAG CCG GCC ATG 205GCC CAG GTG CAG CTA CAG CAG TGG GG OH6S (HuVH4C-SFI)GTC CTC GCA ACT GCG GCC CAG CCG GCC ATG 206GCC CAG STG CAG CTG CAG GAG TCS GG OH7S (HuVH6A-SFI)GTC CTC GCA ACT GCG GCC CAG CCG GCC ATG 207GCC CAG GTA CAG CTG CAG CAG TCA GG OJH1 (HuJH1/2-XHO)GAG TCA TTC TCG ACT CGA GAC RGT GAC CAG 208 GGT GCC OJH2 (HuJH3-XHO)GAG TCA TTC TCG ACT CGA GAC GGT GAC CAT 209 TGT CCC OJH3 (HuJH4/5-XHO)GAG TCA TTC TCG ACT CGA GAC GGT GAC CAG 210 GGT TCC OJH4 (HuJH6-XHO)GAG TCA TTC TCG ACT CGA GAC GGT GAC CGT 211 GGT CCC * Mix in 1:1:1 ratio# Mix in 1:1 ratio + Mix in 1:1 ratio

TABLE 3 Second round VL regions amplification overview 5′ 3′ Share inShare in Template primer primer Product PK/PL(%) Pool VL (%) K1 OK1SOJK1 K1J1 25 PK1 30 OK1S OJK2 K1J2 25 OK1S OJK3 K1J3 10 OK1S OJK4 K1J425 OK1S OJK5 K1J5 15 K2 OK2S OJK1 K2J1 25 PK2 4 OK2S OJK2 K2J2 25 OK2SOJK3 K2J3 10 OK2S OJK4 K2J4 25 OK2S OJK5 K2J5 15 K3 OK3S OJK1 K3J1 25PK3 1 OK3S OJK2 K3J2 25 OK3S OJK3 K3J3 10 OK3S OJK4 K3J4 25 OK3S OJK5K3J5 15 K4 OK4S OJK1 K4J1 25 PK4 19 OK4S OJK2 K4J2 25 OK4S OJK3 K4J3 10OK4S OJK4 K4J4 25 OK4S OJK5 K4J5 15 K5 OK5S OJK1 K5J1 25 PK5 1 OK5S OJK2K5J2 25 OK5S OJK3 K5J3 10 OK5S OJK4 K5J4 25 OK5S OJK5 K5J5 15 K6 OK6SOJK1 K6J1 25 PK6 5 OK6S OJK2 K6J2 25 OK6S OJK3 K6J3 10 OK6S OJK4 K6J4 25OK6S OJK5 K6J5 15 L1 OL1S OJL1 L1J1 30 PL1 14 OL1S OJL2 L1J2 60 OL1SOJL3 L1J3 10 L2 OL2S OJL1 L2J1 30 PL2 10 OL2S OJL2 L2J2 60 OL2S OJL3L2J3 10 L3 OL3S OJL1 L3J1 30 PL3 10 OL3S OJL2 L3J2 60 OL3S OJL3 L3J3 10L4 OL4S OJL1 L4J1 30 PL4 1 OL4S OJL2 L4J2 60 OL4S OJL3 L4J3 10 L5 OL5SOJL1 L5J1 30 PL5 1 OL5S OJL2 L5J2 60 OL5S OJL3 L5J3 10 L6 OL6S OJL1 L6J130 PL6 1 OL6S OJL2 L6J2 60 OL6S OJL3 L6J3 10 L7 OL7S OJL1 L7J1 30 PL7 1OL7S OJL2 L7J2 60 OL7S OJL3 L7J3 10 L8 OL8S OJL1 L8J1 30 PL8 1 OL8S OJL2L8J2 60 OL8S OJL3 L8J3 10 L9 OL9S OJL1 L9J1 30 PL9 1 OL9S OJL2 L9J2 60OL9S OJL3 L9J3 10 VL 100%

TABLE 4 Second round VH regions amplification overview 5′ 3′ Share inShare in Template primer primer Product PK/PL (%) Pool VH (%) H1 OH1SOJH1 H1J1 10 PH1 25 OH1S OJH2 H1J2 10 OH1S OJH3 H1J3 60 OH1S OJH4 H1J420 H2 OH2S OJH1 H2J1 10 PH2 2 OH2S OJH2 H2J2 10 OH2S OJH3 H2J3 60 OH2SOJH4 H2J4 20 H3 OH3S OJH1 H3J1 10 PH3 25 OH3S OJH2 H3J2 10 OH3S OJH3H3J3 60 OH3S OJH4 H3J4 20 H4 OH4S OJH1 H4J1 10 PH4 25 OH4S OJH2 H4J2 10OH4S OJH3 H4J3 60 OH4S OJH4 H4J4 20 H5 OH5S OJH1 H5J1 10 PH5 2 OH5S OJH2H5J2 10 OH5S OJH3 H5J3 60 OH5S OJH4 H5J4 20 H6 OH6S OJH1 H6J1 10 PH6 20OH6S OJH2 H6J2 10 OH6S OJH3 H6J3 60 OH6S OJH4 H6J4 20 H7 OH7S OJH1 H7J110 PH7 1 OH7S OJH2 H7J2 10 OH7S OJH3 H7J3 60 OH7S OJH4 H7J4 20 VH 100%

TABLE 5 Characteristics of the individual IgM memory B cell libraries.IgM memory libraries Cells Libraries Total % Insert PBL % memory Sizefre- % % Donor (×10⁶) B cells (×10⁶) quency ORF Unique Individual 1 3 9674 98 Individual 2 72.5 1.7 5 98 79 98 Individual 3 67.5 1.4 3 96 79 98Individual 4 132.5 2.3 6 98 69 99

TABLE 6 Data of the HA-specific single-chain Fvs. SEQ ID NO SEQ ID NO(nucl (amino acid Name scFv sequence) sequence)* VH-locus VL-locusSC06-141 212 213 VH1 (1-18) VKIV (B3) (Vh 1-115; Vl 132-245) SC06-255115 116 VH1 (1-69) VL1 (V1-16) (Vh 1-121; Vl 138-248) SC06-257 117 118VH1 (1-69) VL2 (V1-4) (Vh 1-121; Vl 138-248) SC06-260 119 120 VH1 (1-69)VL1 (V1-17) (Vh 1-121; Vl 138-248) SC06-261 121 122 VH1 (1-69) VL1(V1-19) (Vh 1-121; Vl 138-249) SC06-262 123 124 VH1 (1-69) VKI (A20) (Vh1-120; Vl 137-245) SC06-268 125 126 VH1 (1-69) VL3 (V2-1) (Vh 1-120; Vl137-243) SC06-272 214 215 VH1 (1-69) VL2 (V1-3) (Vh 1-120; Vl 137-247)SC06-296 216 217 VH1 (1-2) VKIII (A27) (Vh 1-121; Vl 138-246) SC06-301218 219 VH1 (3-23) VKII (A3) (Vh 1-117; Vl 134-246) SC06-307 127 128 VH3(3-21) VKIII (A27) (Vh 1-122; Vl 139-246) SC06-310 129 130 VH1 (1-69)VL3 (V2-14) (Vh 1-121; Vl 138-246) SC06-314 131 132 VH1 (1-69) VL1(V1-17) (Vh 1-121; Vl 138-248) SC06-323 133 134 VH1 (1-69) VKIII (A27)(Vh 1-120; Vl 137-245) SC06-325 135 136 VH1 (1-69) VL2 (V1-4) (Vh 1-121;Vl 138-248) SC06-327 220 221 VH1 (1-69) VL3 (V2-14) (Vh 1-121; Vl138-246) SC06-328 222 223 VH1 (1-69) VKIII (A27) (Vh 1-128; Vl 145-252)SC06-329 224 225 VH1 (1-69) VKIII (A27) (Vh 1-121; Vl 138-246) SC06-331137 138 VH1 (1-69) VL3 (V2-14) (Vh 1-120; Vl 137-245) SC06-332 226 227VH1 (1-69) VKI (A20) (Vh 1-120; Vl 137-243) SC06-334 228 229 VH1 (1-69)VL3 (V2-14) (Vh 1-120; Vl 137-245) SC06-336 230 231 VH1 (1-69) VKIII(A27) (Vh 1-120; Vl 137-245) SC06-339 232 233 VH1 (1-69) VL3 (V2-14) (Vh1-121; Vl 138-246) SC06-342 234 235 VH1 (1-69) VKIV (B3) (Vh 1-126; Vl143-256) SC06-343 236 237 VH1 (1-69) VL3 (V2-14) (Vh 1-120; Vl 137-245)SC06-344 139 140 VH1 (1-69) VL1 (V1-13) (Vh 1-123; Vl 140-250) *betweenbrackets the amino acids making up the heavy chain variable region (VH)and the light chain variable region (VL) is shown

TABLE 7 Data of the CDR regions of the HA specific immunoglobulins. TheSEQ ID NO is given between brackets. Name scFv HCDR1 HCDR2 HCDR3 LCDR1LCDR2 LCDR3 SC06-141 GYYVY (238) WISAYNGNT SRSLDV  KSSQSVLYSS WASTRESQQYYSTPLT NYAQKFQG (240) NNKNYLA (242) (243) (239) (241) SC06-255SYAIS (1) GIIPIFGTTKY HMGYQVRE SGSTFNIGSN SNNQRPS (5) AAWDDILNVAPKFQG (2) TMDV (3) AVD (4) PV (6) SC06-257 SYAIS (1) GIIPIFGTTKYHMGYQVRE TGTSSDVGG EVSNRPS (8) SSYTSSSTYV APKFQG (2) TMDV (3) YNYVS (7)(9) SC06-260 SYAIS (1) GIIPIFGTTKY HMGYQVRE SGSRSNVGD KNTQRPS VAWDDSVDAPKFQG (2) TMDV (3) NSVY (10) (11) GYV (12) SC06-261 SYAIS (1)GIIPIFGTTKY HMGYQVRE SGSSSNIGND DNNKRPS ATWDRRPTA APKFQG (2) TMDV (3)YVS (13) (14) YVV (15) SC06-262 GSAIS (16) GISPLFGTTN GPKYYSEYMRASQGISSYL DASTLRS QRYNSAPPIT YAQKFQG DV (18) A (19) (20) (21) (17)SC06-268 SYAIS (1) GIMGMFGTT SSGYYPEYF SGHKLGDKY QDNRRPS QAWDSSTAVNYAQKFQG QD (23) VS (24) (25) (26) (22) SC06-272 SYAIT (244) GIIGMFGSTNSTGYYPAYL TGTSSDVGG DVSKRPS SSYTSSSTHV YAQNFQG HH (246) YNYVS (247)(248) (249) (245) SC06-296 SYYMH (250) WINPNSGGT EGKWGPQA RASQSVSSSYDASSRAT QQYGSSLW NYAQKFQG AFDI (252) LA (253) (254) (255) (251) SC06-301IYAMS (256) AISSSGDSTY AYGYTFDP RSSQSLLHSN LGSNRAS MQALQTPL YADSVKG(258) GYNYLD (260) (261) (257) (259) SC06-307 SYSMN (27) SISSSSSYIYYGGGSYGAYE RASQRVSSY GASTRAA QQYGRTPLT VDSVKG (28) GFDY (29) LA (30) (31)(32) SC06-310 SYAIS (1) GIIPIFGTTKY HMGYQVRE GGNNIGSKS DDSDRPS QVWDSSSDHAPKFQG (2) TMDV (3) VH (33) (34) AV (35) SC06-314 SYAIS (1) GIIPIFGTTKYHMGYQVRE SGSSSNIGSN RDGQRPS ATWDDNLSG APKFQG (2) TMDV (3) YVY (36) (37)PV (38) SC06-323 SYGIS (39) DIIGMFGSTN SSGYYPAYL RASQSVSSSY GASSRATQQYGSSPRT YAQNFQG PH (41) LA (42) (43) (44) (40) SC06-325 FYSMS (45)GIIPMFGTTN GDKGIYYYY TGTSSDVGG EVSNRPS (8) SSYTSSSTLV YAQKFQG MDV (47)YNYVS (7) (48) (46) SC06-327 THAIS (262) GIIAIFGTAN GSGYHISTPF GGNNIGSKGDDSDRPS QVWDSSSDH YAQKFQG DN (264) VH (265) (266) VV (267) (263)SC06-328 GYAIS (268) GIIPIFGTTNY VKDGYCTLT RASQSVSSSY GASSRAT QQYGSSLTAQKFQG SCPVGWYFD LA (271) (272) (273) (269) L (270) SC06-329 SNSIS (274)GIFALFGTTD GSGYTTRNY RASQSVSSN GASSRAS QQYGSSPLT YAQKFQG FDY (276)YLG (277) (278) (279) (275) SC06-331 SYAIS (1) GIIGMFGTAN GNYYYESSLGGNNIGSKS DDSDRPS QVWDSSSDH YAQKFQG DY (50) VH (33) (34) YV (51) (49)SC06-332 NFAIN (280) GIIAVFGTTK GPHYYSSYM RASQGISTYL AASTLQS QKYNSAPSYAHKFQG DV (282) A (283) (284) (285) (281) SC06-334 SNAVS (286)GILGVFGSPS GPTYYYSYM GGNNIGRNS DDSDRPS QVWHSSSDH YAQKFQG DV (288)VH (289) (290) YV (291) (287) SC06-336 SYAIS (292) GIFGMFGTA SSGYYPQYFRASQSVSSSY GASSRAT QQYGSSSLT NYAQKFQG QD (294) LA (295) (296) (297)(293) SC06-339 SYAIS (298) GIIAIFHTPKY GSTYDFSSGL GGNNIGSKS DDSDRPSQVWDSSSDH AQKFQG DY (300) VH (301) (302) VV (303) (299) SC06-342SYAIS (304) GVIPIFRTAN LNYHDSGTY KSSQSILNSS WASTRES QQYYSSPPT YAQNFQGYNAPRGWFD NNKNYLA (308) (309) (305) P (306) (307) SC06-343 YYAMS (310)GISPMFGTTT SSNYYDSVY GGHNIGSNS DNSDRPS QVWGSSSDH YAQKFQG DY (312)VH (313) (314) YV (315) (311) SC06-344 NYAMS (52) GIIAIFGTPKY IPHYNFGSGSTGSSSNIGAG GNSNRPS GTWDSSLSA AQKFQG (53) YFDY (54) YDVH (55) (56)YV (57)

TABLE 8 Data of the HA-specific IgGs. SEQ ID NO SEQ ID NO SEQ ID NO SEQID NO of nucl. of amino of nucl. of amino sequence acid sequence*sequence acid sequence* Name IgG heavy chain heavy chain light chainlight chain CR6141 316 317 318 319 (Vh 1-115) (Vl 1-114) CR6255 58  5984  85 (Vh 1-121) (Vl 1-111) CR6257 60  61 86  87 (Vh 1-121) (Vl 1-111)CR6260 62  63 88  89 (Vh 1-121) (Vl 1-111) CR6261 64  65 90  91 (Vh1-121) (Vl 1-112) CR6262 66  67 92  93 (Vh 1-120) (Vl 1-109) CR6268 68 69 94  95 (Vh 1-120) (Vl 1-107) CR6272 320 321 322 323 (Vh 1-120) (Vl1-111) CR6296 324 325 326 327 (Vh 1-121) (Vl 1-109) CR6301 328 329 330331 (Vh 1-117) (Vl 1-113) CR6307 70  71 96  97 (Vh 1-122) (Vl 1-108)CR6310 72  73 98  99 (Vh 1-121) (Vl 1-109) CR6314 74  75 100 101 (Vh1-121) (Vl 1-111) CR6323 76  77 102 103 (Vh 1-120) (Vl 1-109) CR6325 78 79 104 105 (Vh 1-121) (Vl 1-111) CR6327 332 333 334 335 (Vh 1-121) (Vl1-109) CR6328 336 337 338 339 (Vh 1-128) (Vl 1-108) CR6329 340 341 342343 (Vh 1-121) (Vl 1-109) CR6331 80  81 106 107 (Vh 1-120) (Vl 1-109)CR6332 344 345 346 347 (Vh 1-120) (Vl 1-107) CR6334 348 349 350 351 (Vh1-120) (Vl 1-109) CR6336 352 353 354 355 (Vh 1-120) (Vl 1-109) CR6339356 357 358 359 (Vh 1-121) (Vl 1-109) CR6342 360 361 362 363 (Vh 1-126)(Vl 1-114) CR6343 364 365 366 367 (Vh 1-120) (Vl 1-109) CR6344 82  83108 109 (Vh 1-123) (Vl 1-111) *between brackets the amino acids makingup the heavy chain variable region (VH) and the light chain variableregion (VL) is shown

TABLE 9 Binding of IgGs to HA (H5N1TV)-expressing PER.C6 ® cells. HA(H5N1TV)-expressing Control PER.C6 ® cells PER.C6 ® cells Anti- 10 1 0.110 1 0.1 body μg/ml μg/ml μg/ml μg/ml μg/ml μg/ml CR6255 414.18 257.1360.43 3.16 2.64 2.48 CR6257 365.17 283.87 62.08 2.59 2.44 2.53 CR6260323.42 168.49 31.06 5.42 3.34 2.57 CR6261 330.77 278.81 85.82 29.4313.22 3.89 CR6262 84.29 20.91 8.06 2.71 2.53 2.48 CR6268 421.70 218.7043.71 3.82 2.74 2.50 CR6307 484.78 266.55 82.42 4.87 3.02 2.48 CR6314399.54 166.98 44.51 5.99 3.49 2.64 CR6323 445.08 116.52 33.38 5.05 2.922.71 CR6325 478.29 239.28 64.36 4.00 3.11 2.57 CR6344 768.25 328.16106.65 80.90 26.33 10.17 CR3014 13.10 10.00 6.21 3.11 2.69 2.55 CR6310*597.04 290.93 86.91 14.92 6.05 4.42 CR6331* 421.14 165.43 41.04 7.874.59 4.55 CR3014* 9.15 7.95 10.51 4.74 4.12 4.57 *Indicates FACSanalyses that were performed in a separate experiment

TABLE 10 Potency of the anti-HA antibodies in the neutralizing antibodytiter assay. Antibody Concentration (μg/ml) CR6255 25 CR6257 12.5 CR626012.5 CR6261 12.5 CR6262 100 CR6268 50 CR6307 50 CR6314 12.5 CR6323 50CR6325 12.5 CR6344 25 CR6310 25 CR6331 100 CR4098 —* *At 50 μg/ml noneutralization was observed

TABLE 11 Potency of the anti-HA antibodies in the neutralizing antibodytiter assay. Antibody Concentration (μg/ml) CR6255 3.12 CR6257 1.56CR6260 3.12 CR6261 0.78 CR6262 25 CR6268 6.25 CR6272 — CR6307 25 CR63106.25 CR6314 3.12 CR6323 6.25 CR6325 6.25 CR6327 6.25 CR6328 25 CR63293.12 CR6331 25 CR6332 12.5 CR6334 6.25 CR6336 25 CR6339 — CR6342 6.25CR6343 50 CR6344 25 — means at 100 μg/ml no neutralization was observed

TABLE 12 Cross-reactivity of anti-H5N1 IgGs to HA molecules of differentHA subtypes as measured by ELISA (OD 492 nm). ND: Not determinedBPL-inact. BPL-inact. A/NC/20/99 NIBRG14 H1 H3 H5 H7 H9 (H1N1) (H5N1)CR6255 1.91 0.08 1.44 0.22 1.97 1.38 1.18 CR6257 1.93 0.08 1.37 0.162.06 1.34 1.21 CR6260 1.88 0.08 1.45 0.19 2.00 1.34 1.17 CR6261 2.040.07 1.44 0.31 2.32 1.46 1.41 CR6262 1.48 0.09 1.02 0.17 1.93 0.51 0.35CR6268 1.78 0.08 1.30 0.15 2.16 1.39 1.13 CR6272 0.81 0.07 0.58 0.150.96 0.92 0.79 CR6307 0.22 0.11 0.99 0.22 0.17 0.23 0.50 CR6310 1.920.08 1.37 0.18 2.17 1.31 1.00 CR6314 1.93 0.07 1.48 0.25 2.21 1.37 1.42CR6323 2.32 0.07 1.89 0.15 2.27 1.40 0.93 CR6325 1.42 0.07 1.30 0.172.04 1.09 1.20 CR6327 1.75 0.08 1.11 0.14 1.93 1.16 0.73 CR6328 2.430.07 1.78 0.16 2.38 1.39 0.98 CR6329 1.98 0.08 1.43 0.17 2.16 1.04 1.01CR6331 1.75 0.06 1.32 0.16 2.02 1.25 0.92 CR6332 2.20 0.15 1.65 0.262.11 1.20 1.11 CR6334 2.04 0.07 1.19 0.15 1.82 1.13 0.91 CR6336 1.920.10 1.41 0.18 2.02 1.09 0.94 CR6339 1.25 0.07 0.81 0.14 1.96 0.94 0.50CR6342 1.99 0.09 1.25 0.17 2.13 1.32 0.90 CR6343 1.28 0.07 0.76 0.151.88 0.91 0.64 CR6344 1.80 0.09 1.31 0.18 2.15 1.31 0.96 CR5111 0.080.07 1.57 0.15 0.17 0.09 0.81 CR3014 0.08 0.07 0.09 0.17 0.16 0.09 0.11No IgG 0.13 0.08 0.09 0.15 0.16 0.09 0.14 Sheep ND ND ND 0.90 2.07 2.622.93 anti-H5

TABLE 13 Epitope mapping of human anti-HA antibodies. CR6261 CR6342CR6307 CR6325 HA1 HA2 CR5111 C179 CR6323 CR6329 H5N1TV TGLRNGVTNKVNSIIDK + + + + Mutant  K---- ------------ + − + + I Mutant  ----------E------ + − + − II Mutant  --M-- ------------ + + + + III Mutant ----- -------R---- + − + + IV Mutant ----- ----------N- + + + + V

TABLE 14 Clinical scores of mice pre-treated with antibody, followed bya lethal challenge with H5N1 influenza virus Number of mice showingclinical signs^(a) Group Ab-dose Study day ID (mg/kg) −1 0 1 2 3 4 5 6 78 9 10 11 12 13 14 15 16 17 18 19 20 21 1 CR6261-15 0 0 0 0 0 0 0 0 0 00 0 0 0 0/9 0/9 0/9 0/9 0/9 0/9 0/9 0/9 0/9 2 CR6261-5 0 0 0 0 0 0 0 0 00 0 2/9 2/9 1/8 0/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7 3 CR6261-2 0 0 0 0 00 0 2 5 5 4/9 3/8 0/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7 4CR6261-0.7 0 0 0 0 0 0 10 10 8/8 7/7 7/7 6/6 4/4 4/4 4/4 4/4 4/4 4/4 4/41/4 1/4 1/4 1/4 5 CR6323-15 0 0 0 0 0 0 1 1 1 1 0/9 0/9 0/9 0/9 0/9 0/90/9 0/9 0/9 0/9 0/9 0/9 0/9 6 CR6325-15 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 00 0 0 0 0 0 0 7 RaH5N3 0 0 0 0 0 0 5 10 10 6/6 1/1 1/1 0/0 0/0 0/0 0/00/0 0/0 0/0 0/0 0/0 0/0 0/0 8 CR3014-15 0 0 0 0 10 10 10 10 9/9 5/5 1/11/1 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 ^(a)out of 10 unlessindicated otherwise

TABLE 15 Respiratory distress of mice pre-treated with antibody,followed by a lethal challenge with H5N1 influenza virus Number of miceshowing respiratory distress^(a) Group Ab-dose Study day ID (mg/kg) −1 01 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 1 CR6261-15 0 0 00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 CR6261-5 0 0 0 0 0 0 0 0 0 0 00 2 1 0 0 0 0 0 0 0 0 0 3 CR6261-2 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 00 0 0 0 4 CR6261-0.7 0 0 0 0 0 0 0 0 8 7 7 6 4 4 4 4 4 4 0 0 0 0 0 5CR6323-15 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 6 CR6325-15 0 00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 7 RaH5N3 0 0 0 0 0 0 0 0 10 61 1 — — — — — — — — — — — 8 CR3014-15 0 0 0 0 0 0 0 10 9 5 1 1 — — — — —— — — — — —

TABLE 16 Clinical scores of mice infected with H5N1 virus and treatedwith antibody at different time points post-infection Time Number ofmice showing clinical signs^(a) Group interval Study day ID p.i. −1 0 12 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 1 4 h 0 0 0 4 4 2 22 0 0 0 0/9 0/9 0/9 0/9 0/9 0/9 0/9 0/9 0/9 0/9 0/9 0/9 2 1 d 0 0 0 1010 10 10 10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 2 d 0 0 0 3 10 10 10 10 8 99 8 5 4 2 0 0 0 0 0 0 0 0 4 3 d 0 0 0 5 5 10 10 10 10 10 10 10 10 3 3 20 0 0 0 0 0 0 5 CR2006 0 0 0 3 7 10 10 10 6/6 1/1 0 0 0 0 0 0 0 0 0 0 00 0 ^(a)with 14 mice in groups 2 and 5 until day 6 p.i.

TABLE 17 Respiratory distress of mice infected with H5N1 virus andtreated with antibody at different time points post-infection TimeNumber of mice showing respiratory distress^(a) Group interval Study dayID p.i. −1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 1 4 h0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 1 d 0 0 0 0 0 0 2 2 0 00 0 0 0 0 0 0 0 0 0 0 0 0 3 2 d 0 0 0 0 0 0 10 10 2 0 0 0 0 0 0 0 0 0 00 0 0 0 4 3 d 0 0 0 0 0 0 10 10 5 9 7 7 4 1 0 0 0 0 0 0 0 0 0 5 CR2006 00 0 0 0 0 10 10 6 1 0 0 0 0 0 0 0 0 0 0 0 0 0 ^(a)score 3

TABLE 18 Mortality of mice infected with H5N1 virus and treated withantibody at different time points post-infection Time Number of livingmice on each study day Group interval Study day ID p.i. −1 0 1 2 3 4 5 67 8 9 10 11 12 13 14 15 16 17 18 19 20 21 1 4 h 10 10 10 10 10 10 10 1010 10 10 9 9 9 9 9 9 9 9 9 9 9 9 2 1 d 10 10 10 10 10 10 10 10 10 10 1010 10 10 10 10 10 10 10 10 10 10 10 3 2 d 10 10 10 10 10 10 10 10 10 1010 10 10 10 10 10 10 10 10 10 10 10 10 4 3 d 10 10 10 10 10 10 10 10 1010 10 10 10 10 10 10 10 10 10 10 10 10 10 5 CR2006 10 10 10 10 10 10 1010 6 1 0 0 0 0 0 0 0 0 0 0 0 0 0

TABLE 19 Mortality of mice infected with H1N1 virus and treated withantibody at different time points prior- and post-infection Time Numberof living mice on each study day interval Study day p.i. −1 0 1 2 3 4 56 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 −1 d  10 10 10 10 10 10 1010 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 1 d 10 10 9 9 9 9 9 9 99 9 9 9 9 9 9 9 9 9 9 9 9 9 2 d 10 10 9 9 9 9 9 9 9 8 8 8 8 8 8 8 8 8 88 8 8 8 3 d 10 10 10 10 10 10 10 9 9 8 8 8 8 8 8 8 8 8 8 8 8 8 8 control10 10 10 10 10 10 10 10 5 2 0 0 0 0 0 0 0 0 0 0 0 0 0

TABLE 20 Clinical scores of mice infected with H1N1 virus and treatedwith antibody at different time points prior- and post-infection TimeNumber of mice showing clinical signs^(a) Group interval Study day IDp.i. −1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 6  d −10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 10 d 1 0 0 0 9 9 9 9 9 0 00 0 0 0 0 0 0 0 0 0 0 0 0 9 d 2 0 0 0 9 9 9 9 9 9 8/8 8/8 0/8 0/8 0/80/8 0/8 0/8 0/8 0/8 0/8 0/8 0/8 0/8 8 d 3 0 0 0 10 10 10 10 9/9 9/9 8/88/8 8/8 8/8 8/8 4/8 3/8 0/8 0/8 0/8 0/8 0/8 0/8 0/8 7 control 0 0 0 1010 10 10 10 5/5 2/2 ^(a)with 9 mice in the 1 d and 2 d p.i. treatmentgroups

TABLE 21 Respiratory distress of mice infected with H1N1 virus andtreated with antibody at different time points prior- and post-infectionTime Number of mice showing respiratory distress^(a) interval Study dayp.i. −1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 −1 0 0 00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 d 0 0 0 4 7 10 2 0 0 0 0 0 0 00 0 0 0 0 0 0 0 0 2 d 0 0 0 7 8 10 9 3 4 2 0 0 0 0 0 0 0 0 0 0 0 0 0 3 d0 0 0 4 10 9 10 9 9 8 8 8 8 6 0 0 0 0 0 0 0 0 0 ctrl 0 0 0 1 9 7 10 10 52 ^(a)a score of 2 or 3

TABLE 22Sequence of HA2 epitope in different influenza subtypes. The underlinedamino acid represents the valine (V) → glutamic acid (E) substitution in the H2N2 escape mutant (mutant II; Table 13) HA2 SEQ ID NO: H5N1:A/Vietnam/1203/2004 GVTNKVNSIIDK 368 H5N1: A/Hong Kong/156/97GVTNKVNSIIDK 369 H5N2: A/mald/PA/84 GVTNKVNSIIDK 368 H1N1: A/PR/8/34GITNKVNSVIEK 372 A/South Carolina/1/1918 A/WSN/33 A/New Caledonia/20/99A/Bangkok/10/83 A/Yamagata/120/86 H2N2: A/Okuda/57 GITNKVNSVIEK 372A/Kumamoto/1/65 A/Korea/426/68 A/Izumi/5/65 H2N2: A/Izumi/5/65 (R)GITNKENSVIEK 373 H6N2: A/Mallard/Netherlands/16/99 GITNKVNSIIDK 374H9N2: A/Hong Kong/1073/99 KITSKVNNIVDK 375 H3N2: A/Aichi/2/68QINGKLNRVIEK 376 H3N2: A/Fukuoka/C29/85 QINGKLNRLIEK 377

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What is claimed is:
 1. An isolated binding molecule able to recognizeand bind to an epitope in the HA2 subunit of the influenza hemagglutininprotein (HA), wherein the binding molecule has neutralizing activityagainst an influenza virus comprising HA of the H5 subtype, and whereinthe binding molecule is able to bind an epitope comprising SEQ IDNO:371.
 2. The binding molecule of claim 1, which also has neutralizingactivity against an influenza virus comprising HA of the H1 subtype. 3.The isolated binding molecule of claim 1, wherein the H5 subtype isselected from the group consisting of H5N1, H5N2, H5N8, and H5N9.
 4. Thebinding molecule of claim 2, wherein the H1 subtype is H1N1.
 5. Thebinding molecule of claim 1, wherein the binding molecule also hasneutralizing activity against an influenza virus comprising HA of theH2, H6, and/or H9 subtype.
 6. An isolated binding molecule able torecognize and bind to an epitope in the HA2 subunit of the influenzahemagglutinin protein (HA), wherein the binding molecule hasneutralizing activity against an influenza virus comprising HA of the H5subtype, and wherein the binding molecule is able to bind an epitopecomprising SEQ ID NO:371, wherein the binding molecule is a humanmonoclonal antibody.
 7. A method of treating and/or prophylaxing againstan influenza infection in a subject, the method comprising:administering the binding molecule of claim 1 to the subject for thetherapeutic treatment and/or prophylactic treatment of influenzainfection in the subject.
 8. The method according to claim 7, whereinthe influenza infection is caused by an influenza virus strainassociated with a pandemic.
 9. The method according to claim 8, whereinthe influenza virus strain is selected from the group consisting ofH1N1, H5N1, H5N2, H5N8, H5N9, an H2-based strain, and H9N2.
 10. Apharmaceutical composition comprising: the binding molecule of claim 1,and a pharmaceutically acceptable excipient.
 11. A method of producing abinding molecule, the method comprising: expressing the binding moleculeof claim 1 from a host comprising a nucleic acid molecule encoding thebinding molecule.
 12. The method of claim 11, further comprising:recovering the expressed binding molecule.
 13. The isolated bindingmolecule of claim 1, which is recombinantly produced.
 14. A compositioncomprising: the isolated binding molecule of claim 1, bound or attachedto a carrier or substrate.
 15. A composition comprising: the isolatedbinding molecule of claim 1, bound to a solid support.
 16. A compositioncomprising: the isolated binding molecule of claim 1, fused to a markersequence.
 17. The composition of claim 16, wherein the marker sequenceis selected from the group consisting of a hexa-histidine tag, ahemagglutinin (HA) tag, a myc tag, and a flag tag.
 18. A compositioncomprising: the isolated binding molecule of claim 1, conjugated to asecond binding molecule.
 19. A composition comprising: the isolatedbinding molecule of claim 1, conjugated to one or more antigens.
 20. Thepharmaceutical composition of claim 10, prepared by a processcomprising: combining the binding molecule with the pharmaceuticallyacceptable excipient to form a mixture; and vacuum drying orlyophilizing the mixture to form a pharmaceutical composition.
 21. Amethod of diagnosing an influenza infection in a subject, the methodcomprising: interacting the binding molecule of claim 1 with abiological sample taken from the subject in order to diagnose influenzainfection in the subject.
 22. The method according to claim 21, whereinthe influenza infection is caused by an influenza virus strainassociated with a pandemic.
 23. The method according to claim 22,wherein the influenza virus strain is selected from the group consistingof H1N1, H5N1, H5N2, H5N8, H5N9, an H2-based strain, and H9N2.